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1.
Viral DNA packaging motors are among the most powerful molecular motors known. A variety of structural, biochemical, and single-molecule biophysical approaches have been used to understand their mechanochemistry. However, packaging initiation has been difficult to analyze because of its transient and highly dynamic nature. Here, we developed a single-molecule fluorescence assay that allowed visualization of packaging initiation and reinitiation in real time and quantification of motor assembly and initiation kinetics. We observed that a single bacteriophage T4 packaging machine can package multiple DNA molecules in bursts of activity separated by long pauses, suggesting that it switches between active and quiescent states. Multiple initiation pathways were discovered including, unexpectedly, direct DNA binding to the capsid portal followed by recruitment of motor subunits. Rapid succession of ATP hydrolysis was essential for efficient initiation. These observations have implications for the evolution of icosahedral viruses and regulation of virus assembly.As part of a virus life cycle, genetic information needs to be incorporated into the newly produced virus particles. Tailed bacteriophages, which probably form the largest biomass of the planet (1), and many eukaryotic viruses such as herpes viruses use powerful ATPase motors to achieve this (2). These motors generate forces as high as 80–100 pN and translocate DNA into a preformed prohead until a DNA condensate of near crystalline density fills the interior (3).The viral packaging motors share a common architecture with the ASCE (additional strand, conserved E) superfamily of multimeric ring ATPases that perform diverse functions such as chromosome segregation (helicases), protein remodeling (chaperones and proteasomes), and cargo transport (dyneins) (4). Although much has been learned about the mechanochemistry of these motors, little is known about how a functional motor is assembled and its activity is initiated. The packaging motors have the difficult task of precisely inserting the end of a viral genome into the capsid at the time of initiation.In a general virus assembly pathway shared by dsDNA viruses, assembly starts at a unique fivefold vertex of the prohead called the portal vertex, which is formed from 12 molecules of the portal protein (5). A protein shell assembles around a protein scaffold and later becomes an empty prohead after the scaffold leaves, or is degraded (6). In most dsDNA bacteriophages as well as herpes viruses a complex of two proteins, known as small and large “terminase” proteins, recognize a specific sequence of DNA in the concatemeric viral genome (e.g., cos site in phage λ and pac site in phage P22) and make a cut to create a dsDNA end (7, 8). The small terminase is responsible for binding to the cos or pac site, whereas the large terminase makes the cut. However, phage phi29 and adenoviruses do not require DNA cutting because the genome is a unit-length molecule with a covalently attached “terminal protein” at the ends (9). The large terminase, which is also an ATPase, then attaches to the protruding end of the portal and assembles into an oligomeric motor that translocates the DNA genome into the empty prohead through the ∼3.5-nm-diameter portal channel using energy from ATP hydrolysis (7, 8). After packaging one unit-length viral genome (headful packaging), the motor dissociates from the full head and the neck and tail proteins assemble on the portal to make an infectious virus.Bacteriophage T4 has been an important model for tailed bacteriophages as well as herpes viruses (10, 11). The T4 packaging motor, a pentamer of gp17 (70 kDa) (large terminase protein) assembled on the gp20 portal dodecamer (12) is the fastest (packaging rate up to ∼2,000 bp/s) of the viral packaging motors studied (13). Gp17 possesses all of the basic enzymatic activities necessary for generating a DNA-full head: ATPase, nuclease, and translocase (14, 15). An oligomeric small terminase protein, gp16, that forms 11-mer and 12-mer rings recognizes the viral genome in vivo, although it lacks strict sequence specificity and is dispensable for packaging in vitro (16). Cryo-EM reconstruction of the packaging motor in complex with the capsid portal, which we will call the “packaging machine,” shows a ring of five gp17 molecules assembled on the prohead portal into a pentameric configuration with the translocation groove facing the channel (12). An electrostatic force-driven translocation mechanism was proposed in which gp17 subunits alternate between the “tensed” (compact) and “relaxed” (extended) conformational states that is coupled to translocation of DNA in a piston-like fashion (12).Genetic and biochemical studies of several packaging systems have delineated the mechanisms of genome recognition and DNA cutting (17, 18). Structural studies (12, 16, 19) and single-molecule optical tweezers (3, 13) and fluorescence spectroscopy (20, 21) approaches have been used to dissect the mechanochemical steps of DNA translocation. However, the transient nature of DNA and protein interactions at the initiation stage, which involve insertion of the dsDNA end into the prohead and triggering of translocation, has been a major challenge (22). The dynamics of motor assembly, timescales of motor–DNA–portal interactions, and mechanism of initiation are poorly understood in any system.Here, we report a single-molecule fluorescence assay that allowed us to dissect packaging initiation starting from a dsDNA end, in real time, by the phage T4 DNA packaging machine. We reconstituted a fully functional minimal T4 packaging complex and imaged individual packaging machines in real time by total internal reflection fluorescence microscopy. Each machine carried out successive DNA translocations and the times for motor assembly and packaging initiation were quantified. Using this assay we found that packaging initiations occurred in bursts with long pauses in between. We discovered that packaging initiation shows unusual plasticity. It can occur through multiple pathways: motor assembly on the portal followed by interaction with DNA and, unexpectedly, direct interaction of DNA with the portal followed by recruitment of the motor subunits. Finally, subtle changes in the ATP binding Walker A P-loop residues that lower the rate of ATP hydrolysis lead to severe defects in packaging initiation. These results provided insights into the dynamics of interactions that lead to single-molecule encapsidation of DNA by a viral packaging machine.  相似文献   

2.
The abundance, genetic diversity, and crucial ecological and evolutionary roles of marine phages have prompted a large number of metagenomic studies. However, obtaining a thorough understanding of marine phages has been hampered by the low number of phage isolates infecting major bacterial groups other than cyanophages and pelagiphages. Therefore, there is an urgent requirement for the isolation of phages that infect abundant marine bacterial groups. In this study, we isolated and characterized HMO-2011, a phage infecting a bacterium of the SAR116 clade, one of the most abundant marine bacterial lineages. HMO-2011, which infects “Candidatus Puniceispirillum marinum” strain IMCC1322, has an ∼55-kb dsDNA genome that harbors many genes with novel features rarely found in cultured organisms, including genes encoding a DNA polymerase with a partial DnaJ central domain and an atypical methanesulfonate monooxygenase. Furthermore, homologs of nearly all HMO-2011 genes were predominantly found in marine metagenomes rather than cultured organisms, suggesting the novelty of HMO-2011 and the prevalence of this phage type in the oceans. A significant number of the viral metagenome sequences obtained from the ocean surface were best assigned to the HMO-2011 genome. The number of reads assigned to HMO-2011 accounted for 10.3%–25.3% of the total reads assigned to viruses in seven viromes from the Pacific and Indian Oceans, making the HMO-2011 genome the most or second-most frequently assigned viral genome. Given its ability to infect the abundant SAR116 clade and its widespread distribution, Puniceispirillum phage HMO-2011 could be an important resource for marine virus research.Viruses are the most abundant biological entities in diverse marine environments, as revealed by electron microscopy, epifluorescence microscopy, and flow cytometry studies (13). The average number of virus-like particles in surface seawater is ∼107 per mL, and it typically exceeds the number of prokaryotes by an order of magnitude. Marine viruses play an important role in nutrient cycles by mediating a significant proportion of bacterial mortality. This so-called “viral shunt” diverts organic matter from particulate forms to dissolved forms, influencing the overall biogeochemistry of various elements (4). Viruses affect the community composition and genetic diversity of marine organisms by selectively infecting susceptible hosts (5, 6), which also increases the genetic diversity of the viruses themselves through mechanisms such as antagonistic coevolution (6, 7).Recent metagenomic studies of the oceans have revealed the numerous novel genetic repertoires of marine viromes (814). However, most genome fragments in these viromes cannot be categorized into any known viral group (8, 9, 13, 14). Because most marine viruses are believed to be phages (15), a more thorough understanding of the ecological and evolutionary roles of marine viruses and a better interpretation of the rapidly increasing virome sequences require the isolation and genomic analysis of individual viruses, especially phages infecting diverse marine bacterioplankton.The importance of phage isolation is exemplified by studies on marine cyanophages and pelagiphages. The isolation and characterization of cyanophages have shown that they have auxiliary metabolic genes (16), including photosynthesis-related genes that are expressed during infection and modulate host metabolism toward successful infection (1721). Cyanophages are important in the diversification of hosts and horizontal gene transfer and are extensively used to interpret marine metagenome sequences (6, 22, 23). Very recently, four pelagiphages, viruses infecting the SAR11 clade, were isolated (24). Pelagiphage genome sequences have been found to be crucial for the interpretation of viromes from the Pacific Ocean (24). However, there are many abundant marine bacterial groups, including SAR86, SAR116, and Bacteroidetes, for which few phages have been isolated or characterized. Given the poor assignment of virome sequences into specific viral groups and the presence of difficult-to-cultivate or unculturable bacterial groups in the ocean, cultivating these bacteria and isolating the phages infecting them are prerequisites for understanding phage diversity.The SAR116 clade is one of the most abundant groups of heterotrophic bacteria inhabiting the surface of the ocean. Since its initial discovery through the cloning of 16S rRNA genes from the Sargasso Sea (25), many culture-independent studies have shown that the SAR116 clade contributes significantly—more than 10% in some cases—to the bacterial assemblages of the marine euphotic zone (2633). The genome sequences of two bacteria in this clade, HIMB100 and “Candidatus Puniceispirillum marinum” IMCC1322, have recently been reported (34, 35). Both strains have genes for proteorhodopsin-based photoheterotrophy, carbon monoxide dehydrogenase, and dimethylsulfoniopropionate demethylase, suggesting the diverse metabolic potential and biogeochemical importance of the SAR116 clade. Considering the widespread distribution and ecologically meaningful genome characteristics of this clade, their phages are likely to be abundant in marine environments and may provide useful references for functional and phylogenetic annotation of marine viromes.Here, we report the isolation and genomic characterization of HMO-2011, a phage that infects strain IMCC1322 of the SAR116 clade. The HMO-2011 genome harbors many novel genes, such as a gene encoding a DNA polymerase with a DnaJ central domain. The genome of HMO-2011 was a notable resource for the identification of unknown marine viral gene fragments, as HMO-2011 accounted for 10.3%–25.3% of viral metagenome reads assigned to viruses.  相似文献   

3.
An approximation to the ∼4-Mbp basic genome shared by 32 strains of Escherichia coli representing six evolutionary groups has been derived and analyzed computationally. A multiple alignment of the 32 complete genome sequences was filtered to remove mobile elements and identify the most reliable ∼90% of the aligned length of each of the resulting 496 basic-genome pairs. Patterns of single base-pair mutations (SNPs) in aligned pairs distinguish clonally inherited regions from regions where either genome has acquired DNA fragments from diverged genomes by homologous recombination since their last common ancestor. Such recombinant transfer is pervasive across the basic genome, mostly between genomes in the same evolutionary group, and generates many unique mosaic patterns. The six least-diverged genome pairs have one or two recombinant transfers of length ∼40–115 kbp (and few if any other transfers), each containing one or more gene clusters known to confer strong selective advantage in some environments. Moderately diverged genome pairs (0.4–1% SNPs) show mosaic patterns of interspersed clonal and recombinant regions of varying lengths throughout the basic genome, whereas more highly diverged pairs within an evolutionary group or pairs between evolutionary groups having >1.3% SNPs have few clonal matches longer than a few kilobase pairs. Many recombinant transfers appear to incorporate fragments of the entering DNA produced by restriction systems of the recipient cell. A simple computational model can closely fit the data. Most recombinant transfers seem likely to be due to generalized transduction by coevolving populations of phages, which could efficiently distribute variability throughout bacterial genomes.The increasing availability of complete genome sequences of many different bacterial and archaeal species, as well as metagenomic sequencing of mixed populations from natural environments, has stimulated theoretical and computational approaches to understand mechanisms of speciation and how prokaryotic species should be defined (18). Much genome analysis and comparison has been at the level of gene content, identifying core genomes (the set of genes found in most or all genomes in a group) and the continually expanding pan-genome. Population genomics of Escherichia coli has been particularly well studied because of its long history in laboratory research and because many pathogenic strains have been isolated and completely sequenced (914). Proposed models of how related groups or species form and evolve include isolation by ecological niche (79, 11, 15), decreased homologous recombination as divergence between isolated populations increases (24, 8, 14, 16), and coevolving phage and bacterial populations (6).E. coli genomes are highly variable, containing an array of phage-related mobile elements integrated at many different sites (17), random insertions of multiple transposable elements (18), and idiosyncratic genome rearrangements that include inversions, translocations, duplications, and deletions. Although E. coli grows by binary cell division, genetic exchange by homologous recombination has come to be recognized as a significant factor in adaptation and genome evolution (9, 10, 19). Of particular interest has been the relative contribution to genome variability of random mutations (single base-pair differences referred to as SNPs) and replacement of genome regions by homologous recombination with fragments imported from other genomes (here referred to as recombinant transfers or transferred regions). Estimates of the rate, extent, and average lengths of recombinant transfers in the core genome vary widely, as do methods for detecting transferred regions and assessing their impact on phylogenetic relationships (1214, 20, 21).In a previous comparison of complete genome sequences of the K-12 reference strain MG1655 and the reconstructed genome of the B strain of Delbrück and Luria referred to here as B-DL, we observed that SNPs are not randomly distributed among 3,620 perfectly matched pairs of coding sequences but rather have two distinct regimes: sharply decreasing numbers of genes having 0, 1, 2, or 3 SNPs, and an abrupt transition to a much broader exponential distribution in which decreasing numbers of genes contain increasing numbers of SNPs from 4 to 102 SNPs per gene (22). Genes in the two regimes of the distribution are interspersed in clusters of variable lengths throughout what we referred to as the basic genome, namely, the ∼4 Mbp shared by the two genomes after eliminating mobile elements. We speculated that genes having 0 to 3 SNPs may primarily have been inherited clonally from the last common ancestor, whereas genes comprising the exponential tail may primarily have been acquired by horizontal transfer from diverged members of the population.The current study was undertaken to extend these observations to a diverse set of 32 completely sequenced E. coli genomes and to analyze how SNP distributions in the basic genome change as a function of evolutionary divergence between the 496 pairs of strains in this set. We have taken a simpler approach than those of Touchon et al. (13), Didelot et al. (14), and McNally et al. (21), who previously analyzed multiple alignments of complete genomes of E. coli strains. The appreciably larger basic genome derived here is not restricted to protein-coding sequences and retains positional information.  相似文献   

4.
Prochlorococcus is an abundant marine cyanobacterium that grows rapidly in the environment and contributes significantly to global primary production. This cyanobacterium coexists with many cyanophages in the oceans, likely aided by resistance to numerous co-occurring phages. Spontaneous resistance occurs frequently in Prochlorococcus and is often accompanied by a pleiotropic fitness cost manifested as either a reduced growth rate or enhanced infection by other phages. Here, we assessed the fate of a number of phage-resistant Prochlorococcus strains, focusing on those with a high fitness cost. We found that phage-resistant strains continued evolving toward an improved growth rate and a narrower resistance range, resulting in lineages with phenotypes intermediate between those of ancestral susceptible wild-type and initial resistant substrains. Changes in growth rate and resistance range often occurred in independent events, leading to a decoupling of the selection pressures acting on these phenotypes. These changes were largely the result of additional, compensatory mutations in noncore genes located in genomic islands, although genetic reversions were also observed. Additionally, a mutator strain was identified. The similarity of the evolutionary pathway followed by multiple independent resistant cultures and clones suggests they undergo a predictable evolutionary pathway. This process serves to increase both genetic diversity and infection permutations in Prochlorococcus populations, further augmenting the complexity of the interaction network between Prochlorococcus and its phages in nature. Last, our findings provide an explanation for the apparent paradox of a multitude of resistant Prochlorococcus cells in nature that are growing close to their maximal intrinsic growth rates.Large bacterial populations are present in the oceans, playing important roles in primary production and the biogeochemical cycling of matter. These bacterial communities are highly diverse (14) yet form stable and reproducible bacterial assemblages under similar environmental conditions (57).These bacteria are present together with high abundances of viruses (phages) that have the potential to infect and kill them (811). Although studied only rarely in marine organisms (1216), this coexistence is likely to be the result of millions of years of coevolution between these antagonistic interacting partners, as has been well documented for other systems (1720). From the perspective of the bacteria, survival entails the selection of cells that are resistant to infection, preventing viral production and enabling the continuation of the cell lineage. Resistance mechanisms include passively acquired spontaneous mutations in cell surface molecules that prevent phage entry into the cell and other mechanisms that actively terminate phage infection intracellularly, such as restriction–modification systems and acquired resistance by CRISPR-Cas systems (21, 22). Mutations in the phage can also occur that circumvent these host defenses and enable the phage to infect the recently emerged resistant bacterium (23).Acquisition of resistance by bacteria is often associated with a fitness cost. This cost is frequently, but not always, manifested as a reduction in growth rate (2427). Recently, an additional type of cost of resistance was identified, that of enhanced infection whereby resistance to one phage leads to greater susceptibility to other phages (14, 15, 28).Over the years, a number of models have been developed to explain coexistence in terms of the above coevolutionary processes and their costs (16, 2932). In the arms race model, repeated cycles of host mutation and virus countermutation occur, leading to increasing breadths of host resistance and viral infectivity. However, experimental evidence generally indicates that such directional arms race dynamics do not continue indefinitely (25, 33, 34). Therefore, models of negative density-dependent fluctuations due to selective trade-offs, such as kill-the-winner, are often invoked (20, 33, 35, 36). In these models, fluctuations are generally considered to occur between rapidly growing competition specialists that are susceptible to infection and more slowly growing resistant strains that are considered defense specialists. Such negative density-dependent fluctuations are also likely to occur between strains that have differences in viral susceptibility ranges, such as those that would result from enhanced infection (30).The above coevolutionary processes are considered to be among the major mechanisms that have led to and maintain diversity within bacterial communities (32, 35, 3739). These processes also influence genetic microdiversity within populations of closely related bacteria. This is especially the case for cell surface-related genes that are often localized to genomic islands (14, 40, 41), regions of high gene content, and gene sequence variability among members of a population. As such, populations in nature display an enormous degree of microdiversity in phage susceptibility regions, potentially leading to an assortment of subpopulations with different ranges of susceptibility to coexisting phages (4, 14, 30, 40).Prochlorococcus is a unicellular cyanobacterium that is the numerically dominant photosynthetic organism in vast oligotrophic expanses of the open oceans, where it contributes significantly to primary production (42, 43). Prochlorococcus consists of a number of distinct ecotypes (4446) that form stable and reproducible population structures (7). These populations coexist in the oceans with tailed double-stranded DNA phage populations that infect them (4749).Previously, we found that resistance to phage infection occurs frequently in two high-light–adapted Prochlorococcus ecotypes through spontaneous mutations in cell surface-related genes (14). These genes are primarily localized to genomic island 4 (ISL4) that displays a high degree of genetic diversity in environmental populations (14, 40). Although about a third of Prochlorococcus-resistant strains had no detectable associated cost, the others came with a cost manifested as either a slower growth rate or enhanced infection by other phages (14). In nature, Prochlorococcus seems to be growing close to its intrinsic maximal growth rate (5052). This raises the question as to the fate of emergent resistant Prochlorococcus lineages in the environment, especially when resistance is accompanied with a high growth rate fitness cost.To begin addressing this question, we investigated the phenotype of Prochlorococcus strains with time after the acquisition of resistance. We found that resistant strains evolved toward an improved growth rate and a reduced resistance range. Whole-genome sequencing and PCR screening of many of these strains revealed that these phenotypic changes were largely due to additional, compensatory mutations, leading to increased genetic diversity. These findings suggest that the oceans are populated with rapidly growing Prochlorococcus cells with varying degrees of resistance and provide an explanation for how a multitude of presumably resistant Prochlorococcus cells are growing close to their maximal known growth rate in nature.  相似文献   

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Many viruses use molecular motors that generate large forces to package DNA to near-crystalline densities inside preformed viral proheads. Besides being a key step in viral assembly, this process is of interest as a model for understanding the physics of charged polymers under tight 3D confinement. A large number of theoretical studies have modeled DNA packaging, and the nature of the molecular dynamics and the forces resisting the tight confinement is a subject of wide debate. Here, we directly measure the packaging of single DNA molecules in bacteriophage phi29 with optical tweezers. Using a new technique in which we stall the motor and restart it after increasing waiting periods, we show that the DNA undergoes nonequilibrium conformational dynamics during packaging. We show that the relaxation time of the confined DNA is >10 min, which is longer than the time to package the viral genome and 60,000 times longer than that of the unconfined DNA in solution. Thus, the confined DNA molecule becomes kinetically constrained on the timescale of packaging, exhibiting glassy dynamics, which slows the motor, causes significant heterogeneity in packaging rates of individual viruses, and explains the frequent pausing observed in DNA translocation. These results support several recent hypotheses proposed based on polymer dynamics simulations and show that packaging cannot be fully understood by quasistatic thermodynamic models.DNA packaging is both a critical step in viral assembly and a unique model for understanding the physics of polymers under strong confinement. Before packaging, the DNA (∼6–60 µm long) forms a loose random coil of diameter ∼1–3 µm. After translocation into the viral prohead (∼50–100 nm in diameter), a ∼10,000-fold volume compaction is achieved. Packaging is driven by a powerful molecular motor that must work against the large forces resisting confinement arising from DNA bending, repulsion between DNA segments, and entropy loss (18).DNA packaging in bacteriophages phi29, lambda, and T4 has been directly measured via single-molecule manipulation with optical tweezers and the packaging motors have been shown to generate forces of >60 pN, among the highest known for biomotors, while translocating DNA at rates ranging from ∼100 bp (for phage phi29, which packages a 19.3-kbp genome into a 42 × 54-nm prohead shell) up to as high as ∼2,000 bp/s (for phage T4, which packages a 171-kbp genome into a 120 × 86-nm prohead) (915). The force resisting packaging rises steeply with prohead filling and has been proposed to play an important role in driving viral DNA ejection (16).Recently, a variety of theoretical models for viral DNA packaging have been proposed (35, 1721). The simplest treat DNA as an elastic rod with repulsive self-interactions and assume that packaging is a quasistatic thermodynamic process, i.e., that the DNA is able to continuously relax to a free-energy minimum state (35, 1921). The DNA arrangement is generally assumed to be an inverse spool with local hexagonal close packing between DNA segments, as suggested by electron microscopy and X-ray scattering studies (22, 23). Such models yield exact analytical predictions that reproduce many of the experimental trends, including the sharp rise in resistance during the latter stages of packaging (35, 20).Dynamic simulations, however, predict differing results. Depending on model and simulation protocol, some predict rapid equilibration into ordered spool or folded toroid conformations, whereas others predict nonequilibrium dynamics and disordered conformations (3, 6, 2431). The packaged DNA conformation also depends on ionic conditions, capsid size and shape, and shape of the internal core structure found in some phages (6, 30). Notably, some electron microscopy studies have also been interpreted as suggesting ordered spooled conformations (22), whereas others have been interpreted as suggesting partly disordered conformations (29). Although some simulations predict nonequilibrium dynamics, several potential caveats are that (i) the DNA has been represented by coarse-grained polymer models with various approximations for physical interactions (6), (ii) the packaging rate used in the simulations is >105 times higher than the measured packaging rate due to computational constraints (3, 2628), and (iii) it has been pointed out by some authors that simulation timescale cannot be directly related to experimental timescale because of the use of coarse-grained models for DNA (25, 28). As noted in early modeling studies, the calculations based on quasistatic models may represent a lower bound on the required packaging forces due to dissipative dynamic losses (4). Whether nonequilibrium dynamics play a significant role in real systems has thus remained an important open question.  相似文献   

8.
Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (14). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (1821), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (2427), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (3037). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.  相似文献   

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Many antagonistic interactions between hosts and their parasites result in coevolution. Although coevolution can drive diversity and specificity within species, it is not known whether coevolutionary dynamics differ among functionally similar species. We present evidence of coevolution within simple communities of Pseudomonas aeruginosa PAO1 and a panel of bacteriophages. Pathogen identity affected coevolutionary dynamics. For five of six phages tested, time-shift assays revealed temporal peaks in bacterial resistance and phage infectivity, consistent with frequency-dependent selection (Red Queen dynamics). Two of the six phages also imposed additional directional selection, resulting in strongly increased resistance ranges over the entire length of the experiment (ca. 60 generations). Cross-resistance to these two phages was very high, independent of the coevolutionary history of the bacteria. We suggest that coevolutionary dynamics are associated with the nature of the receptor used by the phage for infection. Our results shed light on the coevolutionary process in simple communities and have practical application in the control of bacterial pathogens through the evolutionary training of phages, increasing their virulence and efficacy as therapeutics or disinfectants.Many host–parasite associations coevolve, and patterns in this antagonistic interaction are influenced by biology and environment (13). In single-species host–parasite interactions, parasite genotypes show differences in their host ranges and specificities on host genotypes, providing the basis for such coevolution (48). Arms race dynamics (ARD) driven by directional selection favors a broader resistance range in the host against a greater number of parasite genotypes and an increased host range in the parasite allowing more host genotypes to be infected (2, 9). In contrast, fluctuating selection dynamics (FSD), in which there is no directional change in the evolution of the host resistance range, is governed by negative frequency-dependent selection, favoring hosts that resist the most frequently encountered parasite genotypes and parasites that infect the most common host genotypes (911). It has been suggested that ARD predominates during the initial stages of coevolution, when adaptations to the coevolving opponent are largely cost-free, whereas FDS is more significant at later stages, when attack/defense alleles accumulate in the genome and impose costs (12). Thus, when systems shift from ARD to FSD, dynamic coevolutionary equilibria may arise, with constant numbers of attack/defense alleles at the individual level (13) and the continuous frequency-dependent (re)cycling of alleles at the population level (14).Coevolutionary dynamics between hosts and parasites is increasingly investigated using experimental evolution (15) and more specifically time-shift assays (1618), in which hosts or parasites from a given time point are compared with their counterparts from the past or future, enabling the examination of putative reciprocal adaptations (19, 20). Some of the prime experimental models are bacteria and their lytic phages, which exhibit rapid evolution and are amenable to time-shift tests. Recent study indicates that coevolving populations may exhibit either ARD or FSD (12, 2124), and there is some evidence for the genetic mechanism involved [e.g., mutations at tail fiber genes (12, 25)] and for the expansion of the phage host range as coevolution proceeds (6). Most studies of bacteria–phage coevolution involve the model system Pseudomonas fluorescens SBW25 and its lytic phage ϕ2 (15). Although these studies are important for an in-depth understanding of this process, their restriction to a single host–parasite pair is unfortunate, given the immense diversity of bacteria and phages in both terrestrial and aquatic ecosystems (2628) and the importance of many of these organisms in human health (29). Thus, the generality of previous results to other bacterial species and to different phages parasitizing a given bacterium remains an open question.Coevolution may be particularly important in an applied context, namely when predators or parasitic organisms are used for biocontrol (30). For instance, Conrad et al. (31) recently argued that a community perspective with its ecological and evolutionary underpinnings is needed to explore the usefulness of phages as antimicrobial agents to treat cystic fibrosis patients infected with several bacteria and notably strains of Pseudomonas aeruginosa, a congeneric to the model organism P. fluorescens. Different phages naturally have different host ranges (8, 32, 33), but whether phage taxonomic origin influences impacts on P. aeruginosa is not known. Despite the study of phage mixtures to control P. aeruginosa infections (34), the nature of bacterial cross-resistance to phages other than that with which the bacterium evolved has not been addressed.Previous studies are inconclusive regarding whether P. aeruginosa coevolves with bacteriophages (35, 36). Given the ubiquity of this model organism in natural habitats (37) and as a widespread pathogen in hospitals (38, 39), it is important to know whether phage parasitism influences P. aeruginosa population biology and adaptation, whether coevolution between these antagonists actually occurs, and, if so, whether there are general patterns shared by different phages. Here, we test coevolutionary dynamics and their consistency in a panel of lytic bacteriophages and their host P. aeruginosa PAO1. We allowed this bacterium to interact and potentially coevolve with each of six different phage isolates separately, four from the Podoviridae and two from the Myoviridae (Table S1), for a total of 10 serial transfers (∼60 generations). Using bacteria and phages isolated from different time points, we conducted time-shift assays of resistance to infer patterns of the coevolutionary process. Finally, to assess specificity, we performed a cross-resistance assay with evolved bacteria and the six ancestral phage isolates to compare resistance of the bacteria to their “own” phage and to “foreign” phages with which they had not coevolved.  相似文献   

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Filamentous phage are elongated semiflexible ssDNA viruses that infect bacteria. The M13 phage, belonging to the family inoviridae, has a length of ∼1 μm and a diameter of ∼7 nm. Here we present a structural model for the capsid of intact M13 bacteriophage using Rosetta model building guided by structure restraints obtained from magic-angle spinning solid-state NMR experimental data. The C5 subunit symmetry observed in fiber diffraction studies was enforced during model building. The structure consists of stacked pentamers with largely alpha helical subunits containing an N-terminal type II β-turn; there is a rise of 16.6–16.7 Å and a tilt of 36.1–36.6° between consecutive pentamers. The packing of the subunits is stabilized by a repeating hydrophobic stacking pocket; each subunit participates in four pockets by contributing different hydrophobic residues, which are spread along the subunit sequence. Our study provides, to our knowledge, the first magic-angle spinning NMR structure of an intact filamentous virus capsid and further demonstrates the strength of this technique as a method of choice to study noncrystalline, high-molecular-weight molecular assemblies.Filamentous bacteriophage are long, thin, and semiflexible rod viruses that infect bacteria (1, 2). These large assemblies (∼15–35 MDa) contain a circular single-stranded (ss) DNA genome encapsulated in a protein shell. All filamentous phage have a similar life cycle and virion structure despite the relatively high number of strains, with DNA sequence homology varying from almost complete to very little. The unique phage properties make them ideal for a large range of applications such as phage display (3), DNA cloning and sequencing (4, 5), nanomaterial fabrication (68), and as drug-carrying nanomachines (9). In addition, filamentous viruses form a variety of liquid crystals driving the development of both theory and practice of soft-matter physics (10, 11). Filamentous viruses are also associated with various diseases, e.g., CTXϕ phage in cholera toxin (12) and Pf4 phage in cystic fibrosis (13).Phage belonging to the Ff family (M13, fd, f1) are F-pilus–specific viruses that share almost identical genomes and very similar structures. M13 is a 16-MDa virus having a diameter of ∼7 nm and a length of ∼1 μm. The capsid is composed of several thousand identical copies of a major coat protein subunit arranged in a helical array surrounding a core of a circular ssDNA. The major coat proteins constitute ∼85% of the total virion mass, the ssDNA ∼12%, and all other minor proteins (gp3, gp6, gp7, gp9) that are specific for infection and assembly constitute about 3% of the total virion mass (1, 14).Previous structural models for a small number of phages have been obtained by means of X-ray fiber diffraction (1519), static solid-state NMR (20, 21), and cryo-EM (22). Structural models for the Ff family have been proposed based on the three methods; however, satisfactory resolution was only obtained for the Y21M mutant of the fd phage (17, 18, 21) (wt fd is related to M13 by one additional mutation, N12D). The only reported model for M13 (23) (no coordinates available) and models of fd-Y21M from different methods differ in detail (24) and lack accuracy in some structural details such as the N-terminus orientation, the nature of DNA–protein interactions, and sidechain interactions, which are the dominant packing elements of the capsid. The most recent model was built using a combination of static NMR and fiber diffraction (17).According to fiber diffraction, the symmetry of the Ff capsid is C5S2, also referred to as class I symmetry. That is, a fivefold rotation of the major coat protein subunit around the virion axis (pentamers) and an approximate 36° rotation relating two successive pentamers [in fd-Y21M a precise 36° rotation was reported; for fd, values of −33.23° (18) and −34.62° (22) were reported]. All studies report that the coat protein is mostly right-handed, curved, α-helical, with a flexible or disordered N terminus.Magic-angle spinning (MAS) solid-state NMR has become a popular tool for studying the structure and dynamics of biological molecules (2527). The method can be implemented on a variety of systems from small peptides to macromolecular biological assemblies. Integrated approaches can be used to resolve structures of large assemblies (28, 29) and recently, the combination of MAS NMR data, cryo-EM, and Rosetta modeling resulted in a detailed atomic structure of the recombinant type III secretion system needle (30, 31). We have previously performed MAS NMR studies on both wt fd and M13 in a precipitated form (3234). Their chemical shifts pointed to a single homogeneous capsid subunit that is mostly helical and curved with a mobile N terminus. NMR studies of the interactions between the capsid and the DNA reported on the subunit orientation with respect to the viral axis, and indicated that the C terminus undergoes electrostatic interactions with the DNA.In this study, we use homonuclear 2D 13C–13C correlation experiments on sparsely labeled M13 samples together with our prior backbone and sidechain resonance assignments of the M13 phage to acquire MAS NMR structure restraints. Using the CS-Rosetta fold-and-dock protocol (35) we derive an atomic detailed well-converged quaternary structural model of the intact M13 phage viral capsid. The specific bacteriophage symmetry produces four identical, repeating hydrophobic pockets for each subunit, resulting in tight subunit packing that stabilizes the phage assembly.  相似文献   

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Global analysis of gene expression via RNA sequencing was conducted for trisomics for the left arm of chromosome 2 (2L) and compared with the normal genotype. The predominant response of genes on 2L was dosage compensation in that similar expression occurred in the trisomic compared with the diploid control. However, the male and female trisomic/normal expression ratio distributions for 2L genes differed in that females also showed a strong peak of genes with increased expression and males showed a peak of reduced expression relative to the opposite sex. For genes in other autosomal regions, the predominant response to trisomy was reduced expression to the inverse of the altered chromosomal dosage (2/3), but a minor peak of increased expression in females and further reduced expression in males were also found, illustrating a sexual dimorphism for the response to aneuploidy. Moreover, genes with sex-biased expression as revealed by comparing amounts in normal males and females showed responses of greater magnitude to trisomy 2L, suggesting that the genes involved in dosage-sensitive aneuploid effects also influence sex-biased expression. Each autosomal chromosome arm responded to 2L trisomy similarly, but the ratio distributions for X-linked genes were distinct in both sexes, illustrating an X chromosome-specific response to aneuploidy.Changes in chromosomal dosage have long been known to affect the phenotype or viability of an organism (14). Altering the dosage of individual chromosomes typically has a greater impact than varying the whole genome (57). This general rule led to the concept of “genomic balance” in that dosage changes of part of the genome produce a nonoptimal relationship of gene products. The interpretation afforded these observations was that genes on the aneuploid chromosome produce a dosage effect for the amount of gene product present in the cell (8).However, when gene expression studies were conducted on aneuploids, it became known that transacting modulations of gene product amounts were also more prevalent with aneuploidy than with whole-genome changes (914). Assays of enzyme activities, protein, and RNA levels revealed that any one chromosomal segment could modulate in trans the expression of genes throughout the genome (915). These modulations could be positively or negatively correlated with the changed chromosomal segment dosage, but inverse correlations were the most common (1013). For genes on the varied segment, not only were dosage effects observed, but dosage compensation was also observed, which results from a cancelation of gene dosage effects by inverse effects operating simultaneously on the varied genes (9, 10, 1418). This circumstance results in “autosomal” dosage compensation (14, 1618). Studies of trisomic X chromosomes examining selected endogenous genes or global RNA sequencing (RNA-seq) studies illustrate that the inverse effect can also account for sex chromosome dosage compensation in Drosophila (15, 1921). In concert, autosomal genes are largely inversely affected by trisomy of the X chromosome (15, 19, 21).The dosage effects of aneuploidy can be reduced to the action of single genes whose functions tend to be involved in heterogeneous aspects of gene regulation but which have in common membership in macromolecular complexes (8, 2224). This fact led to the hypothesis that genomic imbalance effects result from the altered stoichiometry of subunits that affects the function of the whole and that occurs from partial but not whole-genome dosage change (8, 2225). Genomic balance also affects the evolutionary trajectory of duplicate genes differently based on whether the mode of duplication is partial or whole-genome (22, 23).Here we used RNA-seq to examine global patterns of gene expression in male and female larvae trisomic for the left arm of chromosome 2 (2L). The results demonstrate the strong prevalence of aneuploidy dosage compensation and of transacting inverse effects. Furthermore, because both trisomic males and females could be examined, a sexual dimorphism of the aneuploid response was discovered. Also, the response of the X chromosome to trisomy 2L was found to be distinct from that of the autosomes, illustrating an X chromosome-specific effect. Genes with sex-biased expression, as determined by comparing normal males and females, responded more strongly to trisomy 2L. Collectively, the results illustrate the prevalence of the inverse dosage effect in trisomic Drosophila and suggest that the X chromosome has evolved a distinct response to genomic imbalance as would be expected under the hypothesis that X chromosome dosage compensation uses the inverse dosage effect as part of its mechanism (15).  相似文献   

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We sequenced the genomes of 3,615 strains of serotype Emm protein 1 (M1) group A Streptococcus to unravel the nature and timing of molecular events contributing to the emergence, dissemination, and genetic diversification of an unusually virulent clone that now causes epidemic human infections worldwide. We discovered that the contemporary epidemic clone emerged in stepwise fashion from a precursor cell that first contained the phage encoding an extracellular DNase virulence factor (streptococcal DNase D2, SdaD2) and subsequently acquired the phage encoding the SpeA1 variant of the streptococcal pyrogenic exotoxin A superantigen. The SpeA2 toxin variant evolved from SpeA1 by a single-nucleotide change in the M1 progenitor strain before acquisition by horizontal gene transfer of a large chromosomal region encoding secreted toxins NAD+-glycohydrolase and streptolysin O. Acquisition of this 36-kb region in the early 1980s into just one cell containing the phage-encoded sdaD2 and speA2 genes was the final major molecular event preceding the emergence and rapid intercontinental spread of the contemporary epidemic clone. Thus, we resolve a decades-old controversy about the type and sequence of genomic alterations that produced this explosive epidemic. Analysis of comprehensive, population-based contemporary invasive strains from seven countries identified strong patterns of temporal population structure. Compared with a preepidemic reference strain, the contemporary clone is significantly more virulent in nonhuman primate models of pharyngitis and necrotizing fasciitis. A key finding is that the molecular evolutionary events transpiring in just one bacterial cell ultimately have produced millions of human infections worldwide.Understanding of the nature and timing of many critical molecular events underlying emergence and dissemination of microbial pathogens that cause epidemic disease remains elusive. Comprehensive delineation of the key molecular contributors to these processes is essential for developing better strategies to recognize and predict virulent strain emergence and epidemics, formulate protective public health policies and maneuvers, and develop or modify vaccines. In recent years, fast and inexpensive massively parallel DNA sequencing has facilitated genetically based enhanced understanding of these and other infectious disease problems.Group A Streptococcus (GAS, also known as Streptococcus pyogenes), a Gram-positive bacterial pathogen, causes human infections worldwide (120). For example, GAS is responsible for more than 600 million infections globally each year, including a conservative estimate of 10,000–15,000 severe invasive infections annually in the United States (4). This organism has a long-recognized proclivity to cause epidemic waves, the reasons for which are largely unknown. After several decades of declining incidence, a striking resurgence of severe invasive infections caused by serotype Emm protein 1 (M1) GAS was reported in many countries in the late 1980s and early 1990s (13, 520). The resurgence received widespread public interest in part because of the untimely death of Jim Henson, the Muppeteer, of invasive GAS, and because of severe infections occurring in other notable public figures in Canada and elsewhere (21). This resurgence of severe life-threatening GAS infections was a potent reminder that we know relatively little about the evolutionary genetic events and epidemiological forces that underpin temporal variation in bacterial disease frequency and severity. Lack of precise understanding of these topics significantly hobbles our ability to understand and predict bacterial strain emergence and epidemics.GAS serves as a model pathogen for studying the evolutionary genomics of epidemic disease in part because it causes abundant human infections; comprehensive, population-based strain collections are available from diverse countries; the organism has a relatively small genome size (∼1.8 Mb); and high-quality full-genome sequences are available for strains of diverse M protein serotypes (22, 23). In addition, humans are the pathogen’s only natural host, which means that genetic and epidemiologic events are not obscured by processes occurring in nonhuman hosts or the environment. The primary goal of this study was to test the hypothesis that large-scale genome sequencing and animal infection models would shed new light on heretofore vague molecular events contributing to the evolutionary genomics of epidemic disease in general, and specifically in GAS. Our findings are based on the largest bacterial whole-genome sequencing project reported thus far.  相似文献   

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