Bacteriophage self-counting in the presence of viral replication

When host cells are in low abundance, temperate bacteriophages opt for dormant (lysogenic) infection. Phage lambda implements this strategy by increasing the frequency of lysogeny at higher multiplicity of infection (MOI). However, it remains unclear how the phage reliably counts infecting viral genomes even as their intracellular number increases because of replication. By combining theoretical modeling with single-cell measurements of viral copy number and gene expression, we find that instead of hindering lambda’s decision, replication facilitates it. In a nonreplicating mutant, viral gene expression simply scales with MOI rather than diverging into lytic (virulent) and lysogenic trajectories. A similar pattern is followed during early infection by wild-type phage. However, later in the infection, the modulation of viral replication by the decision genes amplifies the initially modest gene expression differences into divergent trajectories. Replication thus ensures the optimal decision—lysis upon single-phage infection and lysogeny at higher MOI.

Following genome entry into the host cell, temperate bacteriophages must choose between two developmental pathways (1). In the default lytic pathway, rapid viral replication typically culminates in the death of the host cell (lysis) and release of viral progeny. By contrast, in the lysogenic pathway, phages suppress their virulent functions and enter a dormant prophage state (1). To decide on the infected cell’s fate, temperate phages assess the environmental abundance of potential hosts (1, 2). If susceptible host cells are scarce, then producing hundreds of new phages via the lytic pathway would be futile, and, instead, lysogeny should be chosen. To evaluate the relative abundance of viruses and cells, phages use diverse methods. Some achieve this by measuring the number of simultaneously coinfecting phages (multiplicity of infection, MOI) and increasing the frequency of lysogeny at higher MOI (3, 4). Other bacteriophages harness cell–cell communication to assess the frequency of virus–host encounters in their vicinity (5, 6). Notwithstanding the mechanism by which the measurement is performed, a regulatory circuit encoded by the virus must interpret a biological signal reflecting the relative abundance of viruses and host cells and use it to bias a decision between the two possible outcomes of infection.Phage lambda, a temperate bacteriophage that infects Escherichia coli, has long served as the paradigm for viral self-counting (7–10). Direct measurements both in bulk (10, 11) and in single cells (12) demonstrated that a higher number of coinfecting phages increases the probability of lysogeny. Decades of experimental interrogation have resulted in a comprehensive genetic understanding of the virus and the identification of key players involved in the lambda postinfection decision (13–15). However, despite this detailed molecular knowledge of the underlying circuitry, our system-level understanding of how MOI drives the infection outcome is far from complete (12, 16, 17). In contrast to the two-gene “switch” governing lysogenic maintenance (18), the network driving the lysis/lysogeny decision comprises multiple genes, regulating each other through diverse molecular interactions (13). The common theoretical view of the decision is that this genetic network is biased by MOI toward either of two attractors, one corresponding to lytic onset, another to lysogeny (7, 9, 16, 17, 19–22). However, the way this takes place varies between models. Further challenging our ability to decipher the circuit’s function is the fact that, while the eventual gene expression patterns in lysis and lysogeny clearly differ, the initial gene expression cascade following infection appears indistinguishable for both pathways (23).Complicating phages’ task of measuring MOI—and our attempts to decipher how they do it—is the fact that the viral copy number is rapidly increasing inside the infected cell (Fig. 1). Phage replication begins within minutes of genome entry (22) and coincides with the expression of early genes in the decision circuit (23) (Fig. 2). In other words, the initial MOI, which the viral circuitry presumably attempts to measure (24, 25), is soon obfuscated by the presence of additional phage genomes in the cell. Elucidating how lambda succeeds in distinguishing the initial genome number from the instantaneous number present in the cell has remained a challenge partly because of experimental limitations. Within a population, single-cell MOI is broadly distributed (10, 12) (SI Appendix, Fig. S1), necessitating measurements at the level of the individually infected cell. However, simultaneous measurement of the viral copy number and the expression of phage genes has previously not been possible at single-cell resolution.Open in a separate windowFig. 1.The lambda decision circuit measures the MOI even as the viral copy number is changing. A higher MOI increases the propensity to lysogenize. Here, infection by a single lambda phage (Top) results in lysis, whereas coinfection by two phages (Bottom) leads to lysogeny. In choosing cell fate, the infecting phage must respond to the initial number of viral genomes in the cell but ignore the subsequent increase in number because of viral replication.Open in a separate windowFig. 2.A simplified model of the decision network captures the kinetics of mRNA and viral copy number following infection. (A, Top) The three-gene circuit at the heart of the lysis/lysogeny decision. (Bottom) The corresponding segment of the lambda genome. Upon viral entry, P_R expresses both cro and (following a leaky terminator) cII. CII then activates cI expression from P_RE. CI and Cro repress P_R and P_L as well as phage replication. In a lysogen, CI regulates its own expression from P_RM. (B) Images of a single E. coli cell at 10 min following infection by λ cI857 Pam80 P1parS. Phage genomes are labeled using ParB-parS and the mRNA for cI, cro, and cII using single-molecule fluorescence in situ hybridization. The yellow dashed line indicates the cell boundary. (C) The numbers of cI, cro, and cII mRNA per cell at different times following infection by P− phage (λ cI857 Pam80 P1parS) at MOI = 1 to 5. Markers and error bars indicate experimental mean ± SEM per sample (refer to SI Appendix, Table S6 for detailed sample sizes). Solid lines indicate model fit. (D) Viral copy number measured using qPCR following infection at MOI = 1 by P+ and P− phages. Markers and error bars indicate experimental mean ± SD because of qPCR calibration uncertainty. Lines indicate model fits. Refer to SI Appendix for detailed information.Here, we combine single-molecule detection of messenger RNA (mRNA) and phage genomes during infection with mathematical modeling of network dynamics to identify how lambda measures the number of coinfecting phages. To circumvent the complication of a time-varying genome number, we first examined infection by a replication-deficient lambda strain. At various times after infection, we measured, in individual cells, the viral copy number (which, in this case, equals the MOI) and mRNA levels of key lambda genes—cI, cro, and cII. To our surprise, we found no divergence of the mRNA trajectories between low and high MOI, indicative of a transition between the lytic and lysogenic attractors. Instead, gene expression simply scaled with viral dosage. This led us to hypothesize that viral replication is required for obtaining an MOI-dependent lysis/lysogeny decision. To test this hypothesis, we constructed a data-calibrated model for the decision network that included the coupling of phage replication to gene expression. Our model revealed that, indeed, viral replication is inextricably coupled to the lysogeny decision. Early in infection, during a time window set by the dynamics of CII, a short-lived activator, expression of the lysogenic repressor CI scales with MOI, similarly to what we observed in the absence of replication. However, subsequent replication—and its modulation by the decision genes—drive a sharp divergence of cell fates as a function of MOI. Specifically, the initial accumulation of CI at MOI > 1 leads to repression of both cro expression and viral replication, enabling the lysogenic choice. In contrast, at MOI = 1, accumulated CI is insufficient to repress cro expression and replication. Consequently, Cro production from a rapidly increasing number of cro gene copies activates the lytic pathway. We thus find that, rather than hindering lambda’s decision by obscuring the initial MOI, viral replication ensures the appropriate choice of lysis upon infection by a single phage and lysogeny upon coinfection.     

Defensive hypervariable regions confer superinfection exclusion in microviruses

Single-stranded DNA phages of the family Microviridae have fundamentally different evolutionary origins and dynamics than the more frequently studied double-stranded DNA phages. Despite their small size (around 5 kb), which imposes extreme constraints on genomic innovation, they have adapted to become prominent members of viromes in numerous ecosystems and hold a dominant position among viruses in the human gut. We show that multiple, divergent lineages in the family Microviridae have independently become capable of lysogenizing hosts and have convergently developed hypervariable regions in their DNA pilot protein, which is responsible for injecting the phage genome into the host. By creating microviruses with combinations of genomic segments from different phages and infecting Escherichia coli as a model system, we demonstrate that this hypervariable region confers the ability of temperate Microviridae to prevent DNA injection and infection by other microviruses. The DNA pilot protein is present in most microviruses, but has been recruited repeatedly into this additional role as microviruses altered their lifestyle by evolving the ability to integrate in bacterial genomes, which linked their survival to that of their hosts. Our results emphasize that competition between viruses is a considerable and often overlooked source of selective pressure, and by producing similar evolutionary outcomes in distinct lineages, it underlies the prevalence of hypervariable regions in the genomes of microviruses and perhaps beyond.

Numerous studies have found members of the viral family Microviridae as a, if not the, dominating force in the human gut virome (e.g., refs. 1–3). Many human viromes are composed almost entirely of these small, single-stranded DNA (ssDNA) phages, which have fundamentally different evolutionary origins than the more commonly studied double-stranded DNA (dsDNA) phages (4). To date, few aspects of their ecology and evolution as a whole have been studied because most microviruses are known only from metagenomic data rather than from physical isolates (5).Several microviral taxa have been detected as prophages of the Bacteroidetes, Firmicutes, and Proteobacteria (6–9). As prophages, they link their fate to the survival and replication of their bacterial host (10)—“piggybacking-the-winner” rather than “killing-the-winner” by preying on abundant bacteria (11, 12). Therefore, the transition from a lytic to a temperate lifestyle [which, in microviruses, occurred through the acquisition of a short integration motif recognized by bacterial integrase/recombinase systems (9)] imposes new selective pressures on viral populations (13). For example, the lytic microviruses of the subfamily Bullavirinae, exemplified by the venerable phiX174, and the recently discovered temperate members of the subfamily Gokushovirinae both infect Escherichia coli but display very different survival strategies: the former excels at rapid replication and lysis of its bacterial host (14), whereas the latter are slow replicators that can reside in host cultures for long periods of time, with no apparent effect on host fitness (9).Temperate Gokushovirinae, like other prophages, render their bacterial hosts immune to subsequent infection by related viruses (9) via a process referred to as superinfection exclusion or immunity (SiEx) (15–17). SiEx not only serves to defend hosts against further phage infection but also offers an offensive strategy to bacteria. By providing a way to differentiate infected self- from uninfected nonself cells (18), the excision of prophages produces viral particles that infect and kill nonself strains (19, 20). Given the role of SiEx in competition between bacteria harboring different phage types, as well as between the phages themselves, numerous mechanisms of SiEx exist. SiEx has been studied for decades (21, 22) but primarily in double-stranded DNA viruses such as Lambda, P1, T4, and Mu. In these phages, SiEx is typically conferred by accessory proteins that are not essential to viral function and often lost or gained through recombination (e.g., refs. 23–25). However, the small size of microviral genomes and the broadly overlapping gene sets of lytic and temperate microviruses render the evolution of SiEx through the horizontal acquisition of genes unlikely.In this study, we link the evolution of a hypervariable region (HVR) in the DNA pilot protein of microviral capsids to the viral ability to both mediate and overcome SiEx. We demonstrate that multiple divergent variants of this region, resulting from competition between otherwise identical phages, are present in populations of lysogens. By synthesizing hybrid phages composed of segments derived from different gokushoviruses and conducting superinfection experiments, we pinpoint the genomic region responsible for SiEx—a region that is almost uniform in lytic viruses but highly variable in temperate viruses. Our results show that divergent microviruses, as a result of (pro)phage arms races, have converged on the identical defensive strategy based on the evolution of HVRs in the same ancestral, structural gene. These findings advance our understanding of the biology and evolution of the ubiquitous microviruses, whose small genomes lack space to evolve or acquire accessory genes to confer new traits. Rather, already existing genes are repurposed for novel functions while preserving compact genome sizes.     

Contrasted coevolutionary dynamics between a bacterial pathogen and its bacteriophages

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 (1–3). 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 (4–8). 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 (9–11). 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 (16–18), 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, 21–24), 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 (26–28) 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.     

Subdiffusive motion of bacteriophage in mucosal surfaces increases the frequency of bacterial encounters

Bacteriophages (phages) defend mucosal surfaces against bacterial infections. However, their complex interactions with their bacterial hosts and with the mucus-covered epithelium remain mostly unexplored. Our previous work demonstrated that T4 phage with Hoc proteins exposed on their capsid adhered to mucin glycoproteins and protected mucus-producing tissue culture cells in vitro. On this basis, we proposed our bacteriophage adherence to mucus (BAM) model of immunity. Here, to test this model, we developed a microfluidic device (chip) that emulates a mucosal surface experiencing constant fluid flow and mucin secretion dynamics. Using mucus-producing human cells and Escherichia coli in the chip, we observed similar accumulation and persistence of mucus-adherent T4 phage and nonadherent T4∆hoc phage in the mucus. Nevertheless, T4 phage reduced bacterial colonization of the epithelium >4,000-fold compared with T4∆hoc phage. This suggests that phage adherence to mucus increases encounters with bacterial hosts by some other mechanism. Phages are traditionally thought to be completely dependent on normal diffusion, driven by random Brownian motion, for host contact. We demonstrated that T4 phage particles displayed subdiffusive motion in mucus, whereas T4∆hoc particles displayed normal diffusion. Experiments and modeling indicate that subdiffusive motion increases phage–host encounters when bacterial concentration is low. By concentrating phages in an optimal mucus zone, subdiffusion increases their host encounters and antimicrobial action. Our revised BAM model proposes that the fundamental mechanism of mucosal immunity is subdiffusion resulting from adherence to mucus. These findings suggest intriguing possibilities for engineering phages to manipulate and personalize the mucosal microbiome.In all animals, mucosal surfaces provide critical immunological services by both protecting against invading bacterial pathogens and supporting large communities of commensal microorganisms (1, 2). Being exposed to the environment, mucosal surfaces are also the infection sites for many important bacterial diseases, including acute diarrhea and cystic fibrosis in humans. This, combined with their accessibility, make mucosal surfaces attractive venues for phage therapy; that is, the use of bacteriophages (phages) to treat and clear bacterial infections (3, 4). Clinical success so far has been erratic (5). The complexities and dynamics of the mucus layer are rarely considered, and the activity of phages therein is mostly unknown. Not surprisingly, phages effective in vitro do not consistently reduce mucosal bacterial host levels in vivo (6, 7). An understanding of the interactions between phages and their bacterial hosts within the relevant physiological environment is critical for consistent success of phage therapy applications.The multilayered mucus is composed primarily of gel-forming mucin glycoproteins that are continually secreted by the underlying epithelium (8). The mucins self-organize to form a mesh that moves constantly outward from the epithelium, propelled by the ongoing secretion of mucin below (9). As the mucus migrates outward, the mucin concentration decreases and the mesh size (a measure of the distance between neighboring mucin strands) increases. Concurrently, the mucus is degraded by microbial proteases and subjected to shear forces that cause sloughing of the outermost layer (10). Despite this constant outward flux, in a diverse array of animals, phages are enriched on the mucosal surfaces compared with the surrounding milieu (11, 12). Previous in vitro experiments using a T4 phage model (particle size ∼200 nm) demonstrated that this phage adhered to mucus via weak binding between Ig-like domains of the highly antigenic outer capsid protein (Hoc) and the abundant glycans of the mucin glycoproteins (11, 12). Moreover, T4 phage adhering to the mucus layer of tissue culture cells protected the underlying epithelium from bacterial infection. On this basis, we proposed the bacteriophage adherence to mucus (BAM) model, whereby phages adhere to the mucosal surfaces of animals and provide a non-host-derived layer of immunity.     

Pervasive domestication of defective prophages by bacteria

Integrated phages (prophages) are major contributors to the diversity of bacterial gene repertoires. Domestication of their components is thought to have endowed bacteria with molecular systems involved in secretion, defense, warfare, and gene transfer. However, the rates and mechanisms of domestication remain unknown. We used comparative genomics to study the evolution of prophages within the bacterial genome. We identified over 300 vertically inherited prophages within enterobacterial genomes. Some of these elements are very old and might predate the split between Escherichia coli and Salmonella enterica. The size distribution of prophage elements is bimodal, suggestive of rapid prophage inactivation followed by much slower genetic degradation. Accordingly, we observed a pervasive pattern of systematic counterselection of nonsynonymous mutations in prophage genes. Importantly, such patterns of purifying selection are observed not only on accessory regions but also in core phage genes, such as those encoding structural and lysis components. This suggests that bacterial hosts select for phage-associated functions. Several of these conserved prophages have gene repertoires compatible with described functions of adaptive prophage-derived elements such as bacteriocins, killer particles, gene transfer agents, or satellite prophages. We suggest that bacteria frequently domesticate their prophages. Most such domesticated elements end up deleted from the bacterial genome because they are replaced by analogous functions carried by new prophages. This puts the bacterial genome in a state of continuous flux of acquisition and loss of phage-derived adaptive genes.The ubiquity and abundance of bacteriophages (or phages) makes them key actors in bacterial population dynamics (1). Although all phages are able to propagate horizontally between cells, temperate phages also propagate vertically in bacterial (lysogenic) lineages, typically by integrating into the bacterial chromosome as prophages. Very few genes are expressed in the prophage, which replicates with the bacterial chromosome (2). The evolutionary interests of integrated phages (prophages) are partly aligned with those of the host chromosome because rapid proliferation of the latter effectively increases prophage population. Accordingly, prophages protect the host against further phage infection (3), from phagocytosis (4), and provide bacterial pathogens with virulence factors (5). Temperate phages also encode accessory genes that increase host fitness under certain conditions, such as increased growth under nutrient limitation (6), biofilm formation (7), and antibiotic tolerance (8). Some prophages provide bacteria with regulatory switches (9). Functional prophages might also be used as biological weapons by lysogens, because their induction can counteract or delay colonization by nonlysogens (10–12). High diversity and high turnover of temperate phages result in a constant input of new genes in the host genome (13, 14). For example, Escherichia coli prophages contribute to more than 35% of the gene diversity (pangenome) of the species (15). Most temperate phages integrate into a few very specific and conserved integration hot spots in chromosomes and their sequences are adapted to the local frequency of DNA motifs, suggesting adaptation of the phage sequence to the requirements of the prophage state (15).Independently of occasional contributions to bacterial fitness, intact prophages are molecular time bombs that kill their hosts upon activation of the lytic cycle (2). It has been shown that bacterial pseudogenes are under selection for rapid deletion from bacterial genomes (16, 17). Prophage inactivation should be under even stronger selection because these elements can kill the cell. One might thus expect rapid genetic degradation of prophages: either they activate the lytic cycle and kill the cell before accumulating inactivating mutations or they are irreversibly degraded and deleted from the host genome. Bacterial chromosomes have numerous cryptic (defective) prophages and other prophage-derived elements that might result from this evolutionary dynamics (13, 14). Accordingly, functional studies of the full repertoire of prophages of bacterial genomes suggest that the majority of prophages are defective at some level: excision, virion formation, lysis, or infective ability (18, 19).Bacterial genomes encode many molecular systems presumably derived from defective prophages. These include gene transfer agents (GTAs) that transfer random pieces of chromosomal DNA to other cells (20), and bacteriocins and type 6 secretion systems (T6SSs) that are involved in bacterial antagonistic associations (21, 22). Model phage-derived elements, like GTAs or T6SSs, are streamlined and genetically very stable. However, genomes contain a number of elements derived from prophages that fit less neatly in the above categories and perform a number of functions with diverse degrees of efficiency: they parasite other phages, kill other bacteria, or transfer host DNA (23). Finally, prophage-derived structures are also involved in complex animal–bacteria associations (24, 25). These different elements blur the distinction between stable phage-derived elements and prophages ongoing genetic degradation, suggesting that some defective prophages provide adaptive functions to bacteria.Temperate phages and their hosts develop complex antagonistic and mutualistic interactions: depending on the circumstances, prophages can either kill bacteria or increase their fitness. There are no systematic studies of which trend dominates the evolutionary dynamics of prophage–bacteria interactions. Here, we bring to the fore a key related question: how do prophages evolve within the bacterial genome? To answer it, we identified the repertoire of vertically inherited prophages of Escherichia coli and Salmonella enterica. The analysis of these prophages revealed unexpected evolutionary patterns suggesting widespread contribution of prophages to bacterial fitness.     

From the Cover: Genome of a SAR116 bacteriophage shows the prevalence of this phage type in the oceans

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 (1–3). 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 (8–14). 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 (17–21). 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 (26–33). 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.     

Convergent evolution toward an improved growth rate and a reduced resistance range in Prochlorococcus strains resistant to phage

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 (1–4) yet form stable and reproducible bacterial assemblages under similar environmental conditions (5–7).These bacteria are present together with high abundances of viruses (phages) that have the potential to infect and kill them (8–11). Although studied only rarely in marine organisms (12–16), 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 (17–20). 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 (24–27). 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, 29–32). 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, 37–39). 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 (44–46) that form stable and reproducible population structures (7). These populations coexist in the oceans with tailed double-stranded DNA phage populations that infect them (47–49).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 (50–52). 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.     

Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria

The increasing threat of pathogen resistance to antibiotics requires the development of novel antimicrobial strategies. Here we present a proof of concept for a genetic strategy that aims to sensitize bacteria to antibiotics and selectively kill antibiotic-resistant bacteria. We use temperate phages to deliver a functional clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) system into the genome of antibiotic-resistant bacteria. The delivered CRISPR-Cas system destroys both antibiotic resistance-conferring plasmids and genetically modified lytic phages. This linkage between antibiotic sensitization and protection from lytic phages is a key feature of the strategy. It allows programming of lytic phages to kill only antibiotic-resistant bacteria while protecting antibiotic-sensitized bacteria. Phages designed according to this strategy may be used on hospital surfaces and hand sanitizers to facilitate replacement of antibiotic-resistant pathogens with sensitive ones.The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins have evolved in prokaryotes to protect against phage attack and undesired plasmid replication by targeting foreign DNA or RNA (1–3). These systems target nucleic acids, based on short DNA sequences, called spacers, that exist between repeats in the CRISPR array. Transcribed spacers guide Cas proteins to homologous sequences within the foreign nucleic acid, called protospacers, which are subsequently cleaved. The CRISPR-Cas systems have revolutionized molecular biology by providing efficient tools to precisely engineer genomes and manipulate gene expression in various organisms (4–10). CRISPR-Cas systems have also recently been used to phenotypically correct genetic diseases in live animals (11), and their utility is being explored for various therapeutic approaches in mammals. Nevertheless, only limited studies have shown the use of CRISPR-Cas systems to target antibiotic resistance genes or a specific population of virulent bacterial strains (12–17).Two recent elegant studies demonstrated that phage-transferable CRISPR-Cas systems are capable of specifically killing pathogens or resensitizing them to antibiotics (16, 17). These studies, and another study (13), also showed that the transferred CRISPR-Cas system is capable of eliminating specific bacterial populations. Furthermore, they demonstrated that the system might be used against pathogens to effectively treat infected animals. Consequently, it was suggested that the system could be used as a potent antimicrobial agent. Nevertheless, although the results of these studies highlight the potential of a phage-transferable CRISPR-Cas system, the concept of using the system as a direct antimicrobial is similar to conventional phage therapy, which currently faces various obstacles (18). One major obstacle is phage administration into infected tissues; this stems from the phages’ immunogenicity and relative large size compared with antibiotics. One may argue that it would be more efficient to directly kill a pathogen by a lytic phage if it were possible to deliver the CRISPR-Cas–encoding cassette into this pathogen by a phage. Moreover, using the proposed systems in infected patients to resensitize pathogens to antibiotics while antibiotics counterselect for these sensitized pathogens would most likely fail due to escape mutants that are selected by the antibiotics.Here we demonstrate a strategy to counteract the emerging threat of antibiotic-resistant bacteria that evades the above shortcomings. Instead of directly killing the pathogens, we propose to sensitize the pathogens on surfaces or in the human skin flora while concomitantly enriching for these sensitized populations. Patients infected by these antibiotic-sensitive bacteria would thus be treatable by traditional antibiotics. In this strategy, the CRISPR-Cas system is used to destroy specific DNAs that confer antibiotic resistance and to concurrently confer a selective advantage to antibiotic-sensitive bacteria by virtue of resistance to lytic phages. The selective advantage enables to efficiently displace populations of nonsensitized bacteria by killing them with lytic phages. In contrast to conventional phage therapy, this approach does not require administration of phages into the host’s tissues. In addition, it does not aim to directly kill treated bacteria but rather to sensitize them to antibiotics and to kill the nonsensitized bacteria. Therefore, there is no counterselection against the sensitization. The strategy relies on CRISPR spacers that can be rationally designed to target any DNA sequence, including those that encode resistance genes and lytic phages. It thus allows genetically linking a trait that is beneficial to the bacteria (i.e., spacers protecting from lytic phage) with a trait that reverses drug resistance (i.e., spacers targeting resistance genes). The genetic linkage enables selecting antibiotic-sensitized bacterial population by using lytic phages. The integrated construct is designed not only to actively eradicate existing resistance genes but also to eliminate horizontal transfer of these genes between bacteria. Extended use of this technology should thus reduce drug-resistant populations of pathogens on major sources of contamination. Consequently, well-established antibiotics for which resistance currently exists could once again be effective.     

Layering transitions and metastable structures of cholesteric liquid crystals in cylindrical confinement

Our study of cholesteric lyotropic chromonic liquid crystals in cylindrical confinement reveals the topological aspects of cholesteric liquid crystals. The double-twist configurations we observe exhibit discontinuous layering transitions, domain formation, metastability, and chiral point defects as the concentration of chiral dopant is varied. We demonstrate that these distinct layer states can be distinguished by chiral topological invariants. We show that changes in the layer structure give rise to a chiral soliton similar to a toron, comprising a metastable pair of chiral point defects. Through the applicability of the invariants we describe to general systems, our work has broad relevance to the study of chiral materials.

Chiral liquid crystals (LCs) are ubiquitous, useful, and rich systems (1–4). From the first discovery of the liquid crystalline phase to the variety of chiral structures formed by biomolecules (5–9), the twisted structure, breaking both mirror and continuous spatial symmetries, is omnipresent. The unique structure also makes the chiral nematic (cholesteric) LC, an essential material for applications utilizing the tunable, responsive, and periodic modulation of anisotropic properties.The cholesteric is also a popular model system to study the geometry and topology of partially ordered matter. The twisted ground state of the cholesteric is often incompatible with confinement and external fields, exhibiting a large variety of frustrated and metastable director configurations accompanying topological defects. Besides the classic example of cholesterics in a Grandjean−Cano wedge (10, 11), examples include cholesteric droplets (12–16), colloids (17–19), shells (20–22), tori (23, 24), cylinders (25–29), microfabricated structures (30, 31), and films between parallel plates with external fields (32–40). These structures are typically understood using a combination of nematic (achiral) topology (41, 42) and energetic arguments, for example, the highly successful Landau−de Gennes approach (43). However, traditional extensions of the nematic topological approach to cholesterics are known to be conceptually incomplete and difficult to apply in regimes where the system size is comparable to the cholesteric pitch (41, 44).An alternative perspective, chiral topology, can give a deeper understanding of these structures (45–47). In this approach, the key role is played by the twist density, given in terms of the director field n by n⋅∇×n. This choice is not arbitrary; the Frank free energy prefers n⋅∇×n≈−q0=2π/p0 with a helical pitch p0, and, from a geometric perspective, n⋅∇×n≠0 defines a contact structure (48). This allows a number of new integer-valued invariants of chiral textures to be defined (45). A configuration with a single sign of twist is chiral, and two configurations which cannot be connected by a path of chiral configurations are chirally distinct, and hence separated by a chiral energy barrier. Within each chiral class of configuration, additional topological invariants may be defined using methods of contact topology (45–48), such as layer numbers. Changing these chiral topological invariants requires passing through a nonchiral configuration. Cholesterics serve as model systems for the exploration of chirality in ordered media, and the phenomena we describe here—metastability in chiral systems controlled by chiral topological invariants—has applicability to chiral order generally. This, in particular, includes chiral ferromagnets, where, for example, our results on chiral topological invariants apply to highly twisted nontopological Skyrmions (49, 50) (“Skyrmionium”).Our experimental model to explore the chiral topological invariants is the cholesteric phase of lyotropic chromonic LCs (LCLCs). The majority of experimental systems hitherto studied are based on thermotropic LCs with typical elastic and surface-anchoring properties. The aqueous LCLCs exhibiting unusual elastic properties, that is, very small twist modulus K2 and large saddle-splay modulus K24 (51–56), often leading to chiral symmetry breaking of confined achiral LCLCs (53, 54, 56–61), may enable us to access uncharted configurations and defects of topological interests. For instance, in the layer configuration by cholesteric LCLCs doped with chiral molecules, their small K2 provides energetic flexibility to the thickness of the cholesteric layer, that is, the repeating structure where the director n twists by π. The large K24 affords curvature-induced surface interactions in combination with a weak anchoring strength of the lyotropic LCs (62–64).We present a systematic investigation of the director configuration of cholesteric LCLCs confined in cylinders with degenerate planar anchoring, depending on the chiral dopant concentration. We show that the structure of cholesteric configurations is controlled by higher-order chiral topological invariants. We focus on two intriguing phenomena observed in cylindrically confined cholesterics. First, the cylindrical symmetry renders multiple local minima to the energy landscape and induces discontinuous increase of twist angles, that is, a layering transition, upon the dopant concentration increase. Additionally, the director configurations of local minima coexist as metastable domains with point-like defects between them. We demonstrate that a chiral layer number invariant distinguishes these configurations, protects the distinct layer configurations (45), and explains the existence of the topological defect where the invariant changes.     

Structural changes in bacteriophage T7 upon receptor-induced genome ejection

Many tailed bacteriophages assemble ejection proteins and a portal–tail complex at a unique vertex of the capsid. The ejection proteins form a transenvelope channel extending the portal–tail channel for the delivery of genomic DNA in cell infection. Here, we report the structure of the mature bacteriophage T7, including the ejection proteins, as well as the structures of the full and empty T7 particles in complex with their cell receptor lipopolysaccharide. Our near–atomic-resolution reconstruction shows that the ejection proteins in the mature T7 assemble into a core, which comprises a fourfold gene product 16 (gp16) ring, an eightfold gp15 ring, and a putative eightfold gp14 ring. The gp15 and gp16 are mainly composed of helix bundles, and gp16 harbors a lytic transglycosylase domain for degrading the bacterial peptidoglycan layer. When interacting with the lipopolysaccharide, the T7 tail nozzle opens. Six copies of gp14 anchor to the tail nozzle, extending the nozzle across the lipopolysaccharide lipid bilayer. The structures of gp15 and gp16 in the mature T7 suggest that they should undergo remarkable conformational changes to form the transenvelope channel. Hydrophobic α-helices were observed in gp16 but not in gp15, suggesting that gp15 forms the channel in the hydrophilic periplasm and gp16 forms the channel in the cytoplasmic membrane.

Many double-stranded DNA (dsDNA) viruses, including tailed bacteriophages and herpesviruses, have a portal attached to a unique pentameric vertex of their icosahedral capsid shell (1–3). The portal is a dodecameric channel for viral DNA packaging and ejection. The tailed bacteriophages and herpesviruses encapsidate DNA in the capsid shell through the portal channel (4–10), and the last packaged DNA is held by tunnel loops (or β-hairpins for herpesviruses) in the portal (11–16). The last packaged DNA in most of the tailed bacteriophages and herpesvirus is the first to be ejected during the genome delivery (17). In tailed bacteriophages, the portal connects to a tail, which serves to recognize host cell receptors and deliver the genome into the cytoplasm (18). Gram-negative bacteriophage in Podoviridae initiate infection through a specific interaction of its receptor-binding protein with the receptor lipopolysaccharide (LPS) on the host cell surface. The phages in Podoviridae have a noncontractile tail that is too short to span the gram-negative bacteria envelope that comprises the outer membrane, the cytoplasmic membrane, and the peptidoglycan layer in the hydrophilic periplasm in between (19). After adsorption, a signal is transmitted for the release of internal ejection proteins to form a channel that extends the tail across the cell envelope and that allows for subsequent genome ejection into the infected cell (20–23). In many previous studies, structural analyses have been performed at resolutions of 9 to 40 Å on this highly coordinated dynamic infection process (21–26). These studies have provided insights on structural changes of phage particles that accompany the infection steps before and after the genome ejection. However, these studies did not resolve structures of the internal ejection proteins. Furthermore, the relative low resolutions cannot clarify the dynamic genome ejection process orchestrated by the ejection proteins, portal, and tail.Escherichia coli bacteriophage T7, a member of the Podoviridae family, has been used as a model for understanding the DNA packaging and delivery mechanism that are common to tailed phages and related dsDNA viruses (10, 21, 27–33). T7 has an icosahedral capsid shell formed by gene product 10 (gp10). The 12-fold portal (gp8) shares a very similar topology with those in other phages and herpesviurses (14–16, 30, 34). The tail comprises a 12-fold adaptor protein gp11 assembly, a sixfold nozzle protein gp12 assembly, and six subunits of trimeric tail fiber gp17 (21, 30). These tail fibers are responsible for bacterial receptor recognition and adsorption (21, 33). On top of the portal within the capsid shell is a hollow cylinder-shaped core structure (10, 28) formed by the ejection proteins (core proteins) gp14, gp15, and gp16, which have been suggested to form a transenvelope channel for the genome delivery into the infected cell (20, 35, 36). The gp16 harbors lytic transglycosylase (LTase) activity, which allows for penetration into the bacterial peptidoglycan layer (37).In this study, we present the structure of the mature bacteriophage T7 with internal core proteins at near-atomic resolution and the structures of the full and empty T7 particles in complex with their cell receptor at subnanometer and near-atomic resolutions, respectively. Our reconstruction reveals that the core in the mature T7 is formed by a fourfold gp16 ring, an eightfold gp15 ring, and a putative eightfold gp14 ring. The putative gp14 structures mediate the core–portal interaction. The gp15 and gp16 are mainly composed of helix bundles, and gp16 harbors a LTase domain. When the T7 phage interacts with the LPS, the tail nozzle opens. Six copies of gp14 anchor to the sixfold tail channel, extending the tail across the LPS lipid bilayer. A conformational change in the portal then triggers the genome ejection. Our structures reveal the structural changes of the phage genome-delivery molecular machines after the genome delivery.     

α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation

Physiologically, α-synuclein chaperones soluble NSF attachment protein receptor (SNARE) complex assembly and may also perform other functions; pathologically, in contrast, α-synuclein misfolds into neurotoxic aggregates that mediate neurodegeneration and propagate between neurons. In neurons, α-synuclein exists in an equilibrium between cytosolic and membrane-bound states. Cytosolic α-synuclein appears to be natively unfolded, whereas membrane-bound α-synuclein adopts an α-helical conformation. Although the majority of studies showed that cytosolic α-synuclein is monomeric, it is unknown whether membrane-bound α-synuclein is also monomeric, and whether chaperoning of SNARE complex assembly by α-synuclein involves its cytosolic or membrane-bound state. Here, we show using chemical cross-linking and fluorescence resonance energy transfer (FRET) that α-synuclein multimerizes into large homomeric complexes upon membrane binding. The FRET experiments indicated that the multimers of membrane-bound α-synuclein exhibit defined intermolecular contacts, suggesting an ordered array. Moreover, we demonstrate that α-synuclein promotes SNARE complex assembly at the presynaptic plasma membrane in its multimeric membrane-bound state, but not in its monomeric cytosolic state. Our data delineate a folding pathway for α-synuclein that ranges from a monomeric, natively unfolded form in cytosol to a physiologically functional, multimeric form upon membrane binding, and show that only the latter but not the former acts as a SNARE complex chaperone at the presynaptic terminal, and may protect against neurodegeneration.α-Synuclein is an abundant presynaptic protein that physiologically acts to promote soluble NSF attachment protein receptor (SNARE) complex assembly in vitro and in vivo (1–3). Point mutations in α-synuclein (A30P, E46K, H50Q, G51D, and A53T) as well as α-synuclein gene duplications and triplications produce early-onset Parkinson''s disease (PD) (4–10). Moreover, α-synuclein is a major component of intracellular protein aggregates called Lewy bodies, which are pathological hallmarks of neurodegenerative disorders such as PD, Lewy body dementia, and multiple system atrophy (11–14). Strikingly, neurotoxic α-synuclein aggregates propagate between neurons during neurodegeneration, suggesting that such α-synuclein aggregates are not only intrinsically neurotoxic but also nucleate additional fibrillization (15–18).α-Synuclein is highly concentrated in presynaptic terminals where α-synuclein exists in an equilibrium between a soluble and a membrane-bound state, and is associated with synaptic vesicles (19–22). The labile association of α-synuclein with membranes (23, 24) suggests that binding of α-synuclein to synaptic vesicles, and its dissociation from these vesicles, may regulate its physiological function. Membrane-bound α-synuclein assumes an α-helical conformation (25–32), whereas cytosolic α-synuclein is natively unfolded and monomeric (refs. 25, 26, 31, and 32; however, see refs. 33 and 34 and Discussion for a divergent view). Membrane binding by α-synuclein is likely physiologically important because in in vitro experiments, α-synuclein remodels membranes (35, 36), influences lipid packing (37, 38), and induces vesicle clustering (39). Moreover, membranes were found to be important for the neuropathological effects of α-synuclein (40–44).However, the relation of membrane binding to the in vivo function of α-synuclein remains unexplored, and it is unknown whether α-synuclein binds to membranes as a monomer or oligomer. Thus, in the present study we have investigated the nature of the membrane-bound state of α-synuclein and its relation to its physiological function in SNARE complex assembly. We found that soluble monomeric α-synuclein assembles into higher-order multimers upon membrane binding and that membrane binding of α-synuclein is required for its physiological activity in promoting SNARE complex assembly at the synapse.     

Collapse of cooperation in evolving games

Game theory provides a quantitative framework for analyzing the behavior of rational agents. The Iterated Prisoner’s Dilemma in particular has become a standard model for studying cooperation and cheating, with cooperation often emerging as a robust outcome in evolving populations. Here we extend evolutionary game theory by allowing players’ payoffs as well as their strategies to evolve in response to selection on heritable mutations. In nature, many organisms engage in mutually beneficial interactions and individuals may seek to change the ratio of risk to reward for cooperation by altering the resources they commit to cooperative interactions. To study this, we construct a general framework for the coevolution of strategies and payoffs in arbitrary iterated games. We show that, when there is a tradeoff between the benefits and costs of cooperation, coevolution often leads to a dramatic loss of cooperation in the Iterated Prisoner’s Dilemma. The collapse of cooperation is so extreme that the average payoff in a population can decline even as the potential reward for mutual cooperation increases. Depending upon the form of tradeoffs, evolution may even move away from the Iterated Prisoner’s Dilemma game altogether. Our work offers a new perspective on the Prisoner’s Dilemma and its predictions for cooperation in natural populations; and it provides a general framework to understand the coevolution of strategies and payoffs in iterated interactions.Iterated games provide a framework for studying social interactions (1–6) that allows researchers to address pervasive biological problems such as the evolution of cooperation and cheating (2, 7–12). Simple examples such as the Iterated Prisoner’s Dilemma, Snowdrift, and Stag Hunt games (13–18) showcase a startling array of counterintuitive social behaviors, especially when studied in a population replicating under natural selection (16, 19–25). Despite the subject’s long history, a systematic treatment of all evolutionary robust cooperative outcomes for even the simple Iterated Prisoner’s Dilemma has only recently emerged (21, 26–29).Understanding the evolution of strategies in a population under fixed payoffs already poses a steep challenge. To complicate matters further, in many biological settings the payoffs themselves may also depend on the genotypes of the players. Changes to the payoff matrix have been studied in a number of contexts, including one-shot two-player games (13), payoff evolution without strategy evolution (30, 31), under environmental “shocks” to the payoff matrix (32–34), and using continuous games (22, 23, 35). Here we adopt a different approach, and we explicitly study the coevolutionary dynamics between strategies and payoffs in iterated two-player games. We decouple strategy mutations from payoff mutations, and we leverage results on the evolutionary robustness of memory-1 strategies with arbitrary payoff matrices to explore the relationship between payoff evolution and the prevalence of cooperation in a population. We identify a feedback between the costs and benefits of cooperation and the evolutionary robustness of cooperative strategies. Depending on the functional form (35) of the relationship between costs and benefits, this feedback may either reinforce the evolutionary success of cooperation or else precipitate its collapse. In particular, we show that cooperation will always collapse when there are diminishing returns for mutual cooperation.     

Sulfur radical species form gold deposits on Earth

Current models of the formation and distribution of gold deposits on Earth are based on the long-standing paradigm that hydrogen sulfide and chloride are the ligands responsible for gold mobilization and precipitation by fluids across the lithosphere. Here we challenge this view by demonstrating, using in situ X-ray absorption spectroscopy and solubility measurements, coupled with molecular dynamics and thermodynamic simulations, that sulfur radical species, such as the trisulfur ion S3−, form very stable and soluble complexes with Au⁺ in aqueous solution at elevated temperatures (>250 °C) and pressures (>100 bar). These species enable extraction, transport, and focused precipitation of gold by sulfur-rich fluids 10–100 times more efficiently than sulfide and chloride only. As a result, S3− exerts an important control on the source, concentration, and distribution of gold in its major economic deposits from magmatic, hydrothermal, and metamorphic settings. The growth and decay of S3− during the fluid generation and evolution is one of the key factors that determine the fate of gold in the lithosphere.The formation of gold deposits on Earth requires aqueous fluids that extract gold from minerals and magmas and transport and precipitate the metal as economic concentrations in ores that are three to six orders of magnitude larger than the Au mean content (∼0.001 ppm) of common crustal and mantle rocks (1–9). However, natural data on gold contents in fluids are very scarce due to difficulties of direct access to deep geothermal fluid samples, rarity of representative fluid inclusions trapped in minerals, and analytical limitations for this chemically most inert metal (1, 4, 9, 10). The paucity of direct data makes it difficult to quantify the capacity of the fluids to transport gold and the factors controlling the sources, formation, and distribution of the economic resources of gold and associated metals across the lithosphere. Thus, knowledge of gold speciation and solubility in the fluid phase is required.Terrestrial hydrothermal fluids systematically contain sulfur and chloride—compounds that have long been known to favor gold dissolution in aqueous solution (e.g., refs. 11 and 12). Following this common knowledge, the interpretation of gold transfers across the lithosphere has been based on the fundamental assumption that only hydrogen sulfide (HS⁻) and chloride (Cl⁻) can form stable complexes with aurous gold, Au⁺, which is the main gold oxidation state in hydrothermal fluids (1–6, 11–15). Among these species, aurous bis(hydrogen sulfide), Au(HS)2−, and dichloride, AuCl2−, have long been regarded as the major carriers of gold in hydrothermal fluids, depending on temperature (T), pressure (P), acidity (pH), redox potential (f_O2), and salt and sulfur concentrations (1, 5, 6, 13, 15). In addition, other minor hydroxide, chloride, and sulfide species (AuOH, AuCl, AuHS) have also been tentatively suggested in some studies to account for the low Au solubility (typically part-per-billion level, ppb) measured in dilute S- and Cl-poor experimental solutions (e.g., refs. 6 and 15). Most available data suggest that the sulfide complexes attain significant concentrations (>1 ppm Au) only in H₂S-rich neutral-to-alkaline (pH > 6–7) solutions at low-to-moderate temperatures (<250–300 °C), whereas the chloride complexes contribute to Au solubility only in highly acidic (pH < 3) chloride-rich (typically >10 wt% NaCl equivalent) and strongly oxidizing [above the oxygen fugacity of the hematite–magnetite (HM) buffer] solutions above 300 °C.In between these two contrasting hydrothermal solution compositions lies a vast domain of geological fluids, which are commonly generated by magma degassing at depth (2, 3, 9) or prograde metamorphism of sedimentary rocks at high temperatures (7, 8, 10). These fluids are characterized by variable salt content, slightly acidic to neutral pH, and the presence of both oxidized (sulfate and sulfur dioxide) and reduced (H₂S) sulfur forms in a wide temperature range (∼300–700 °C). Predicted concentrations of the gold sulfide and chloride species in such fluids are generally rather low (<0.1–1.0 ppm) to account for a number of enigmatic features of gold geochemistry such as the existence of large deposits without relation to magmatic plutons in metamorphic and sedimentary rocks (e.g., Carlin-type and orogenic) implying deep, likely mantle-derived, Au-rich sources, the observation of highly anomalous Au grades (up to thousands of parts per million) in hydrothermal veins, and huge variations (more than three orders of magnitude) of the ratio of Au to other metals (e.g., Cu, Ag, Mo) in ores (1–4, 6–10, 16). These fluids, which have created the major part of economic gold resources on Earth (1–4), may carry much higher Au concentrations, of tens to hundreds of parts per million, as reported from rare fluid inclusion analyses (4, 6, 9, 10) and a few laboratory experiments of Au solubility (17–20). How gold is transported by such fluids remains, however, controversial, and a variety of other species with H₂S, Cl, As, and alkali metal ligands (3, 14, 17–20) or Au nanoparticles (4, 6, 12) were suggested. Thus, a consistent picture of Au speciation and transport in deep and hot crustal fluids is lacking, hampering our understanding of geochemical fluxes of gold across the lithosphere and the formation of gold economic resources.In particular, all existing Au speciation models ignore sulfur radical species such as the trisulfur ion S3−, which is ubiquitous in chemical and engineering products (21) and was recently shown to be stable in the aqueous fluid phase over a wide temperature (T from 200 °C to ∼700 °C) and pressure (P from saturated vapor pressure to ∼30 kbar) range (22–24). The omission of S3− in current models of hydrothermal fluids is due to its very rapid breakdown to sulfate and sulfide in aqueous solution upon cooling, which prohibits the detection of S3− in experimental and natural fluid (and melt) samples brought to ambient conditions. It is thus only recently that the abundance and thermodynamic stability of this important sulfur species could be systematically characterized at high T−P using in situ Raman spectroscopy (22–24). These studies showed that significant amounts of trisulfur ion (>10–100 ppm) may be reached in fluids typical of magmatic and metamorphic environments, which are characterized by elevated dissolved S concentrations (>1,000 ppm), slightly acidic to neutral pH (between ∼3 and 7), and redox conditions enabling coexistence of sulfate (or sulfur dioxide) and hydrogen sulfide.To quantify the effect of S3− on Au behavior in hydrothermal fluids, here we combined in situ X-ray absorption spectroscopy (XAS) and hydrothermal reactor measurements with first-principles molecular dynamics (FPMD) and thermodynamic modeling of Au local atomic structure and solubility in aqueous solutions saturated with gold metal and containing hydrogen sulfide, sulfate and S3− (SI Appendix). These solutions are representative of fluids that formed major types of gold deposits in the crust: 200–500 °C, 300–1,000 bar, 0.1–3.0 wt% S, 3 < pH < 8, and oxygen fugacity f_O2 between the nickel−nickel oxide (NNO) and HM buffer (1–4).     

From the Cover: Asymmetric cryo-EM structure of the canonical Allolevivirus Qβ reveals a single maturation protein and the genomic ssRNA in situ

Single-stranded (ss) RNA viruses infect all domains of life. To date, for most ssRNA virions, only the structures of the capsids and their associated protein components have been resolved to high resolution. Qβ, an ssRNA phage specific for the conjugative F-pilus, has a T = 3 icosahedral lattice of coat proteins assembled around its 4,217 nucleotides of genomic RNA (gRNA). In the mature virion, the maturation protein, A₂, binds to the gRNA and is required for adsorption to the F-pilus. Here, we report the cryo-electron microscopy (cryo-EM) structures of Qβ with and without symmetry applied. The icosahedral structure, at 3.7-Å resolution, resolves loops not previously seen in the published X-ray structure, whereas the asymmetric structure, at 7-Å resolution, reveals A₂ and the gRNA. A₂ contains a bundle of α-helices and replaces one dimer of coat proteins at a twofold axis. The helix bundle binds gRNA, causing denser packing of RNA in its proximity, which asymmetrically expands the surrounding coat protein shell to potentially facilitate RNA release during infection. We observe a fixed pattern of gRNA organization among all viral particles, with the major and minor grooves of RNA helices clearly visible. A single layer of RNA directly contacts every copy of the coat protein, with one-third of the interactions occurring at operator-like RNA hairpins. These RNA–coat interactions stabilize the tertiary structure of gRNA within the virion, which could further provide a roadmap for capsid assembly.Single-stranded (ss) RNA viruses are an abundant type of virus and infect all domains of life (1–4). One of the best-studied ssRNA virus systems is the Leviviridae, which infects Gram-negative bacteria via a variety of retractile pili (5); extensive genetic and biochemical studies have been performed on two of these phages: MS2 and Qβ (5–11). All of the Leviviridae have the same core genome, spanning 3.4–4.3 kb, encoding the maturation protein, the coat protein, and a subunit of RNA-dependent RNA replicase (SI Appendix, Fig. S1) (10). The MS2-like phages, designated true leviviruses, have a fourth gene that encodes the lysis protein, whereas the Qβ-like phages, designated alloleviviruses, have the lysis function as an additional feature of the maturation protein (called A₂ in Qβ). Qβ also encodes a minor coat protein, called A₁, arising from occasional read-through of the stop codon of the major coat protein; it has been estimated that the A₁ protein replaces 3–10 copies of the major coat protein in the virion (11) and is required for infection (12). A₁ consists of a coat domain and a read-through domain separated by a flexible linker (13). Unlike most dsDNA phages, which use specialized protein machinery to pump their genomic DNA into a capsid preassembled around a protein scaffold (14–17), ssRNA viruses, including the Leviviridae, assemble their coat proteins around the genomic RNA (gRNA), presumably because the extremely small genome does not allow for genes to encode proteins that help package the genetic material. Therefore, ssRNA viruses require direct gRNA–coat protein interactions, some of which are specific, to self-assemble the virion (18). This raises interesting questions, including how the viral RNA is selectively encapsidated over host RNAs. Addressing these questions will lead to a better understanding of the physiology of ssRNA viruses, and potentially novel therapeutics against them (19).In Leviviridae, both Qβ and MS2 have capsids with T = 3 morphology, meaning each coat protein monomer adopts one of three conformers, termed A, B, and C (20, 21). Within the viral capsid, conformers A and B form an asymmetric A/B dimer and two C conformers form a symmetric C/C dimer. The gRNAs of Qβ and MS2 form secondary structures both in vitro (22, 23) and in vivo (7). There is a specific RNA hairpin near the start of the replicase gene called the “operator,” which has a high binding affinity for the coat protein (24, 25). Although both MS2 and Qβ operators form hairpins, the coat proteins for each phage are selectively attracted to their specific operator to the point at which, if purified RNAs from both phages are mixed with the coat protein of just one phage, only the cognate RNA is encapsidated (8). In MS2, the maturation protein binds to specific regions on the 5′ and 3′ of the gRNA during capsid assembly (26).Neither the maturation protein nor the gRNA of Leviviridae was resolved in the previous high-resolution structural studies by X-ray crystallography, most likely because of the asymmetry of these two components in the stacking of symmetric particles within the crystals (20, 21). The first glimpse of RNA within the capsid came from a low-resolution structure of MS2 using single-particle cryo-EM (27). On the basis of another single-particle cryo-EM structure with icosahedral symmetry applied, it was proposed that MS2 has two concentric shells of RNA within the phage capsid (28). Subsequent work, using cryo-electron tomography and subtomogram averaging, yielded a 39-Å resolution symmetry-free density map of MS2 (29), suggesting the gRNA adopts the same conformation for each virus particle within the capsid and the maturation protein replaces a C/C dimer of the coat proteins. However, at this low resolution, it was not clear how the maturation protein, which contributes ∼1% of the molecular mass of the entire virion, interacts with the rest of the capsid or how the gRNA is organized inside the capsid shell.In this study, we report the cryo-EM structures of the canonical Allolevivirus Qβ with and without symmetry applied, at 3.7- and 7-Å resolutions, respectively. Our structures reveal features never seen before for Qβ, such as the structure of A₂, symmetry deviation of the coat proteins, organization of gRNA, and the interactions between them. These results are discussed in terms of a model for viral assembly and gRNA release in Qβ.     

The virota and its transkingdom interactions in the healthy infant gut

Virome and 16/18S analyses were performed on 304 longitudinal fecal samples of eight infants. The gut virota—the collection of all viruses present in the gut—was dominated by bacteriophages, which were nearly absent at birth and emerged rapidly within the first weeks after birth. Over 85% of phage reads correspond to 305 near-complete genomes, most of which (70.5%) were individual infant–specific, including two crAssphages, whereas 7.8% of phages were present in at least 50% of infants. Bacterial hosts could be predicted for 80% of phages, mainly infecting Firmicutes. Strong temporal correlations between phages and their predicted bacterial hosts were identified for >40% of our phages, and together with the observation of a decreasing fraction of phages with a temperate lifestyle further suggest that phages are induced from early-colonizing bacteria. The vast majority (>86%) of identified eukaryotic viruses, known to cause gastroenteritis, occurred without clinical signs, and an increase in the rate of infection occurred after day-care entrance. On average, 112 genomic contigs of distinct anelloviruses could be identified per infant, some of which were shed at >1 y. The identified plant viruses reflected the infant diet. Finally, the sporadic identification of fungi and parasites argues against the presence of such stable communities in the study population. Overall, this work provides a very high temporal resolution on how the different members of the infant gut microbiota, and especially the virome, develop over time in the gut of healthy infants, and might serve as valuable baseline knowledge for further studies investigating the effect of perturbations in the infant gut microbiota.

The human gut microbiota is a complex ecosystem, harboring members of several kingdoms of life, including animalia (parasites), fungi, protists, archaeabacteria, and bacteria, as well as viruses infecting all of these kingdoms in addition to the human host. All these complex interactions play an important role in health and disease (1). The bacterial component is by far the most studied and has been shown to be highly temporally stable in healthy adults (2, 3). Infants start their lives with a gut that is largely sterile (4), and prokaryotes colonize their intestinal tract in a stepwise manner (5, 6), until a stable adult-like composition is reached by the age of approximately 2 y (7). Disturbances in this primary colonization process can result in life-long health consequences and have been associated with a broad range of diseases, such as inflammatory bowel disease (IBD) (8), asthma (9), and type I diabetes (T1D) (10).The assessment of the role of the viral component of the gut microbiota (i.e., gut virota), through the analyses of their collective genomes (i.e., the virome), in health and disease is lagging behind, but more and more associations with human diseases such as IBD (11, 12), T1D (13), and colorectal cancer (14) are being made. In terms of composition, adult and infant gut virota are dominated by bacteriophages (15–18), most of which have remained unidentified and are therefore referred to as “viral dark matter” (19, 20), complicating virome analyses. Members of different eukaryotic viral families (Adenoviridae, Anelloviridae, Astroviridae, Parvoviridae, Picornaviridae, and Reoviridae) have been described in healthy infant stool samples and, although most of them can cause human infections and disease, they are often observed in the absence of clinical signs (21, 22). Although evidence for a beneficial role of eukaryotic gut viruses in health is scarce, experiments in mice have shown a potential role of enteric viruses in the development of normal intestinal morphology and function (23). One of the most prevalent viral families of the eukaryotic virota in healthy infants is the family Anelloviridae (22, 24). This viral family has not been associated with any disease so far and a beneficial role of these viruses in human health has been suggested (25).The role of bacteriophages in the gut virota and their link with human health remain understudied but, due to the transkingdom interactions between bacteriophages and their bacterial hosts, they are assumed to be crucial in shaping bacterial communities (26). Furthermore, their interaction dynamics remain unresolved and can only be unraveled in dense longitudinal datasets. The few studies on the virome in healthy infants and adults suggest a striking similarity to what was observed for prokaryotes: going from a (nearly) sterile composition at birth (18) toward an adult composition with a high temporal stability (15, 17, 27). Shkoporov and colleagues showed, in addition to temporal stability, also a strong individuality (17) in 10 adults sampled monthly for a period of 12 mo. Furthermore, they also conclude that phage populations are not showing classical lytic kill-the-winner dynamics in adults but rather a behavior consistent with a temperate phage lifestyle. The first longitudinal virome study in multiple healthy infants by Lim and colleagues suggested that, in contrast to increasing bacteriome diversity and richness over time, the highest richness, diversity, and abundance of phages were observed in the first months of life, followed by a significant decrease over time (22). The richness of eukaryotic viruses increased with age, suggesting that they are derived from environmental exposure (22). A recent study by Liang and colleagues investigated the infant gut virome at three time points after birth (month 0, 1, and 4) and observed a stepwise colonization of the infant gut. Samples from month 1 are dominated by phage particles induced from prophages integrated in pioneering bacteria and, in samples from month 4, eukaryotic viruses infecting human cells become more prominent (18).For the eukaryotic component of the human gut microbiome (i.e., Eukaryota), even less is known. For particular fungi and parasites, a causal role in disease is well-appreciated (28). However, increasingly, these organisms are also considered to be commensals in the human gut and are even used in (probiotics) treatments for several diseases (29, 30). In healthy adults, the gut fungal composition [mainly composed of the phyla Ascomycota and Basiodiomycota (31)] seems very variable among individuals and over time (32, 33). Protists that have already been identified in healthy human stool samples include members of the Blastocystis, Entamoeba, and Trichomonas (34, 35). In infants, eukaryome abundance profiles have, in contrast to the stable stepwise colonization of prokaryotes, been shown to be unstable in early life (36). The lack of successional patterns and a stable presence of these eukaryotes in the infant gut led to the suggestion that in early life no stable eukaryotic community is formed (36). Some researchers even argue that such stable communities of gut eukaryotes are never formed and that they are only passengers via oral and dietary sources (37, 38).In this study, we provide an in-depth analysis of gut microbiome dynamics, with a focus on the virome, in healthy infants during the first year of life. To study the virome, metagenomic sequencing was performed on purified virus-like particles (VLPs) of 304 samples from 8 infants (on average, 38 samples per infant), making this the most densely sampled healthy infant gut virome dataset to date. Most studies only look at a small fraction of their virome data, largely ignoring viral dark matter (19, 20). Using an elaborative bioinformatics viral characterization approach focusing not only on similarity-based methods but also including characterization at functional and specific genome structure levels (such as k-mer usage) allowed us to strongly reduce the amount of viral dark matter. To investigate transkingdom interactions, we overlaid our findings with the previously published 16S ribosomal RNA (rRNA) gene bacterial composition of these same samples (6). Furthermore, we also characterized the presence of eukaryotes using 18S rRNA gene sequencing. This study is unique because it studies gut community assembly across all kingdoms of life with a high temporal resolution.     

Activity-based photoacoustic probe for biopsy-free assessment of copper in murine models of Wilson’s disease and liver metastasis

The development of high-performance photoacoustic (PA) probes that can monitor disease biomarkers in deep tissue has the potential to replace invasive medical procedures such as a biopsy. However, such probes must be optimized for in vivo performance and exhibit an exceptional safety profile. In this study, we have developed PACu-1, a PA probe designed for biopsy-free assessment (BFA) of hepatic Cu via photoacoustic imaging. PACu-1 features a Cu(I)-responsive trigger appended to an aza-BODIPY dye platform that has been optimized for ratiometric sensing. Owing to its excellent performance, we were able to detect basal levels of Cu in healthy wild-type mice as well as elevated Cu in a Wilson’s disease model and in a liver metastasis model. To showcase the potential impact of PACu-1 for BFA, we conducted two blind studies in which we were able to successfully identify Wilson’s disease animals from healthy control mice in each instance.

Photoacoustic (PA) imaging is a light-in, sound-out technique that has emerged as a promising biomedical approach for the noninvasive assessment of various ailments in humans, ranging from arthritis to cancer (1, 2). Excitation of an endogenous pigment such as hemoglobin in blood or melanin in tissue can provide contrast, since relaxation via nonradiative decay can trigger thermoelastic expansion of the surrounding tissue. Repeatedly irradiating a region of interest with a pulsed laser can result in pressure waves that can be readily detected by ultrasound transducers. Since ultrasound at clinically relevant frequencies can travel through the body with minimal perturbation, it is possible to accurately pinpoint the source of the signal to afford high-resolution images at centimeter imaging depths (3). Beyond label-free applications, the utility of PA imaging for disease detection has been augmented by the recent development of acoustogenic probes (activatable PA probes) that give an off-on signal enhancement or ratiometric readout (4, 5). Such examples, as well as imaging agents for other modalities, are designed based on the principles of activity-based sensing, which leverages the chemical reactivity of an analyte to probe and manipulate the system under investigation (6–8). Notable acoustogenic molecules include those that can visualize dysregulated enzymatic activities (9–12), properties of the disease tissue microenvironment (13–15), as well as small molecule– and metal ion–based disease biomarkers (16–24). However, replacing an invasive medical procedure, such as a liver biopsy, with an acoustogenic probe is an immense challenge, since the in vivo performance and safety profile of such a chemical tool must be exceptional. Thus, despite undesirable shortcomings, such as the potential to develop severe infections, false negatives due to collection of nondiseased tissue, and the inability to directly monitor disease progression in real time (25), liver biopsies are still commonly employed to assess biomarkers in conditions such as Wilson’s disease (WD) (26) and cancer (27).It is noteworthy that elevated levels of hepatic copper (Cu) are a common biomarker shared by these conditions. In WD, Cu accumulates in the liver due to a genetic mutation in the Cu exporter, ATP7B, and this can lead to chronic liver damage, which can become fatal if not treated (28, 29). In the context of cancer, Cu is elevated in many solid tumors including breast (30, 31) and lung (32, 33) cancers, which generally metastasize to the liver. Since Cu can promote angiogenesis and drive tumor progression, biopsy-free assessment (BFA) of Cu in metastatic lesions is critical. A number of Cu probes exists for various modalities (34, 35), including examples compatible with in vivo applications via fluorescent (36) and bioluminescent (37) imaging. However, approaches that involve the emission of photons are more suitable for shallow imaging depths (millimeter range) owing to scattering and attenuation of light. More recently, our group (16, 38) and others (39) have developed Cu probes for PA imaging to achieve greater tissue penetration and higher resolution. However, these probes are designed to target Cu(II), whereas intracellular Cu exists predominantly in the +1 form owing to the highly reducing environment of the cell (40). To overcome this challenge, we present the development of PACu-1, an acoustogenic probe for Cu(I), and its application in BFA of hepatic Cu in a WD model and a liver metastasis model. Moreover, we designed two unbiased BFA blind studies to identify WD mice from healthy wild-type (WT) controls using PACu-1.     

The NMR–Rosetta capsid model of M13 bacteriophage reveals a quadrupled hydrophobic packing epitope

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 (6–8), 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 (15–19), 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 (25–27). 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 (32–34). 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.     

Structures and topological defects in pressure-driven lyotropic chromonic liquid crystals

Lyotropic chromonic liquid crystals are water-based materials composed of self-assembled cylindrical aggregates. Their behavior under flow is poorly understood, and quantitatively resolving the optical retardance of the flowing liquid crystal has so far been limited by the imaging speed of current polarization-resolved imaging techniques. Here, we employ a single-shot quantitative polarization imaging method, termed polarized shearing interference microscopy, to quantify the spatial distribution and the dynamics of the structures emerging in nematic disodium cromoglycate solutions in a microfluidic channel. We show that pure-twist disclination loops nucleate in the bulk flow over a range of shear rates. These loops are elongated in the flow direction and exhibit a constant aspect ratio that is governed by the nonnegligible splay-bend anisotropy at the loop boundary. The size of the loops is set by the balance between nucleation forces and annihilation forces acting on the disclination. The fluctuations of the pure-twist disclination loops reflect the tumbling character of nematic disodium cromoglycate. Our study, including experiment, simulation, and scaling analysis, provides a comprehensive understanding of the structure and dynamics of pressure-driven lyotropic chromonic liquid crystals and might open new routes for using these materials to control assembly and flow of biological systems or particles in microfluidic devices.

Lyotropic chromonic liquid crystals (LCLCs) are aqueous dispersions of organic disk-like molecules that self-assemble into cylindrical aggregates, which form nematic or columnar liquid crystal phases under appropriate conditions of concentration and temperature (1–6). These materials have gained increasing attention in both fundamental and applied research over the past decade, due to their distinct structural properties and biocompatibility (4, 7–14). Used as a replacement for isotropic fluids in microfluidic devices, nematic LCLCs have been employed to control the behavior of bacteria and colloids (13, 15–20).Nematic liquid crystals form topological defects under flow, which gives rise to complex dynamical structures that have been extensively studied in thermotropic liquid crystals (TLCs) and liquid crystal polymers (LCPs) (21–29). In contrast to lyotropic liquid crystals that are dispersed in a solvent and whose phase can be tuned by either concentration or temperature, TLCs do not need a solvent to possess a liquid-crystalline state and their phase depends only on temperature (30). Most TLCs are shear-aligned nematics, in which the director evolves toward an equilibrium out-of-plane polar angle. Defects nucleate beyond a critical Ericksen number due to the irreconcilable alignment of the directors from surface anchoring and shear alignment in the bulk flow (24, 31–33). With an increase in shear rate, the defect type can transition from π-walls (domain walls that separate regions whose director orientation differs by an angle of π) to ordered disclinations and to a disordered chaotic regime (34). Recent efforts have aimed to tune and control the defect structures by understanding the relation between the selection of topological defect types and the flow field in flowing TLCs. Strategies to do so include tuning the geometry of microfluidic channels, inducing defect nucleation through the introduction of isotropic phases or designing inhomogeneities in the surface anchoring (35–39). LCPs are typically tumbling nematics for which α2α3 < 0, where α2 and α3 are the Leslie viscosities. This leads to a nonzero viscous torque for any orientation of the director, which allows the director to rotate in the shear plane (22, 29, 30, 40). The tumbling character of LCPs facilitates the nucleation of singular topological defects (22, 40). Moreover, the molecular rotational relaxation times of LCPs are longer than those of TLCs, and they can exceed the timescales imposed by the shear rate. As a result, the rheological behavior of LCPs is governed not only by spatial gradients of the director field from the Frank elasticity, but also by changes in the molecular order parameter (25, 41–43). With increasing shear rate, topological defects in LCPs have been shown to transition from disclinations to rolling cells and to worm-like patterns (25, 26, 43).Topological defects occurring in the flow of nematic LCLCs have so far received much more limited attention (44, 45). At rest, LCLCs exhibit unique properties distinct from those of TLCs and LCPs (1, 2, 4–6, 44). In particular, LCLCs have significant elastic anisotropy compared to TLCs; the twist Frank elastic constant, K2, is much smaller than the splay and bend Frank elastic constants, K1 and K3. The resulting relative ease with which twist deformations can occur can lead to a spontaneous symmetry breaking and the emergence of chiral structures in static LCLCs under spatial confinement, despite the achiral nature of the molecules (4, 46–51). When driven out of equilibrium by an imposed flow, the average director field of LCLCs has been reported to align predominantly along the shear direction under strong shear but to reorient to an alignment perpendicular to the shear direction below a critical shear rate (52–54). A recent study has revealed a variety of complex textures that emerge in simple shear flow in the nematic LCLC disodium cromoglycate (DSCG) (44). The tumbling nature of this liquid crystal leads to enhanced sensitivity to shear rate. At shear rates γ˙<1 s−1, the director realigns perpendicular to the flow direction adapting a so-called log-rolling state characteristic of tumbling nematics. For 1 s−1<γ˙<10 s−1, polydomain textures form due to the nucleation of pure-twist disclination loops, for which the rotation vector is parallel to the loop normal, and mixed wedge-twist disclination loops, for which the rotation vector is perpendicular to the loop normal (44, 55). Above γ˙>10 s−1, the disclination loops gradually transform into periodic stripes in which the director aligns predominantly along the flow direction (44).Here, we report on the structure and dynamics of topological defects occurring in the pressure-driven flow of nematic DSCG. A quantitative evaluation of such dynamics has so far remained challenging, in particular for fast flow velocities, due to the slow image acquisition rate of current quantitative polarization-resolved imaging techniques. Quantitative polarization imaging traditionally relies on three commonly used techniques: fluorescence confocal polarization microscopy, polarizing optical microscopy, and LC-Polscope imaging. Fluorescence confocal polarization microscopy can provide accurate maps of birefringence and orientation angle, but the fluorescent labeling may perturb the flow properties (56). Polarizing optical microscopy requires a mechanical rotation of the polarizers and multiple measurements, which severely limits the imaging speed. LC-Polscope, an extension of conventional polarization optical microscopy, utilizes liquid crystal universal compensators to replace the compensator used in conventional polarization microscopes (57). This leads to an enhanced imaging speed and better compensation for polarization artifacts of the optical system. The need for multiple measurements to quantify retardance, however, still limits the acquisition rate of LC-Polscopes.We overcome these challenges by using a single-shot quantitative polarization microscopy technique, termed polarized shearing interference microscopy (PSIM). PSIM combines circular polarization light excitation with off-axis shearing interferometry detection. Using a custom polarization retrieval algorithm, we achieve single-shot mapping of the retardance, which allows us to reach imaging speeds that are limited only by the camera frame rate while preserving a large field-of-view and micrometer spatial resolution. We provide a brief discussion of the optical design of PSIM in Materials and Methods; further details of the measurement accuracy and imaging performance of PSIM are reported in ref. 58.Using a combination of experiments, numerical simulations and scaling analysis, we show that in the pressure-driven flow of nematic DSCG solutions in a microfluidic channel, pure-twist disclination loops emerge for a certain range of shear rates. These loops are elongated in the flow with a fixed aspect ratio. We demonstrate that the disclination loops nucleate at the boundary between regions where the director aligns predominantly along the flow direction close to the channel walls and regions where the director aligns predominantly perpendicular to the flow direction in the center of the channel. The large elastic stresses of the director gradient at the boundary are then released by the formation of disclination loops. We show that both the characteristic size and the fluctuations of the pure-twist disclination loops can be tuned by controlling the flow rate.