Primate phageomes are structured by superhost phylogeny and environment

Humans harbor diverse communities of microorganisms, the majority of which are bacteria in the gastrointestinal tract. These gut bacterial communities in turn host diverse bacteriophage (hereafter phage) communities that have a major impact on their structure, function, and, ultimately, human health. However, the evolutionary and ecological origins of these human-associated phage communities are poorly understood. To address this question, we examined fecal phageomes of 23 wild nonhuman primate taxa, including multiple representatives of all the major primate radiations. We find relatives of the majority of human-associated phages in wild primates. Primate taxa have distinct phageome compositions that exhibit a clear phylosymbiotic signal, and phage–superhost codivergence is often detected for individual phages. Within species, neighboring social groups harbor compositionally and evolutionarily distinct phageomes, which are structured by superhost social behavior. Captive nonhuman primate phageome composition is intermediate between that of their wild counterparts and humans. Phage phylogenies reveal replacement of wild great ape–associated phages with human-associated ones in captivity and, surprisingly, show no signal for the persistence of wild-associated phages in captivity. Together, our results suggest that potentially labile primate-phage associations have persisted across millions of years of evolution. Across primates, these phylosymbiotic and sometimes codiverging phage communities are shaped by transmission between groupmates through grooming and are dramatically modified when primates are moved into captivity.

Mammals harbor diverse communities of microorganisms, the majority of which are bacteria in the gastrointestinal tract. Gut bacterial communities in turn host diverse phage communities that influence their structure, function, colonization patterns, and ultimately superhost health [the superhost is the host for bacteria that in turn host the phages (1)]. For example, enriched phage communities in human intestinal mucus can act as an acquired immune system by limiting mucosal bacterial populations (2), while dysbiotic gut phageomes are associated with health conditions such as type II diabetes (3), colitis (4), and stunting (5). Transplantation of healthy viral filtrates restored health in Clostridioides difficile patients (6), while in vitro studies suggest phages from stunted children shape bacterial populations differently from those of healthy children (5), supporting a direct link between phageome composition and disease. However, despite their importance in gut microbial ecosystems, the ecological and evolutionary processes that gave rise to these communities remain poorly resolved. Recent work on the widespread crAssphage suggests it might demonstrate long-term associations with its superhosts (7), similar to patterns described for many bacteria (8, 9).Primates host distinct bacterial communities, such that more phylogenetically related host taxa have more similar gut microbial composition (8, 10). The structure of these communities thus recapitulates the host phylogeny [i.e., phylosymbiosis (8, 10)], potentially reflecting widespread cospeciation of bacteria and hosts or phylogenetic conservation of the environments that shape bacterial communities (8, 9). Such long-term host–bacterial associations would imply restricted transmission of bacterial lineages within—rather than between—host lineages (8). This pattern of transmission may be facilitated by the tendency for primates to live in organized societies (11), creating opportunities for bacterial transmission to conspecifics (12, 13). When removed from their natural social and ecological environments and placed in captivity, primates quickly develop humanized bacterial microbiomes (14, 15). This apparent plasticity makes the long-term associations of primates with particular bacterial lineages all the more striking (8, 9).Here, we investigate whether these key findings about primate-associated gut bacterial communities can be generalized to phages. We explore drivers of phage community composition and individual phage lineage evolution in primate superhosts across multiple scales and environments, with a particular emphasis on the potential role of social transmission. We then examine the phageomes of captive primates to understand the flexibility of phage communities in response to the environment and the potential of phage transmission between superhosts. Lastly, we explore whether temperate versus virulent phage lifestyles influence the observed patterns in phage community composition and/or individual phage lineage evolution.     

A molecular link between cell wall biosynthesis,translation fidelity,and stringent response in Streptococcus pneumoniae

Survival in the human host requires bacteria to respond to unfavorable conditions. In the important Gram-positive pathogen Streptococcus pneumoniae, cell wall biosynthesis proteins MurM and MurN are tRNA-dependent amino acyl transferases which lead to the production of branched muropeptides. We demonstrate that wild-type cells experience optimal growth under mildly acidic stressed conditions, but ΔmurMN strain displays growth arrest and extensive lysis. Furthermore, these stress conditions compromise the efficiency with which alanyl-tRNA^Ala synthetase can avoid noncognate mischarging of tRNA^Ala with serine, which is toxic to cells. The observed growth defects are rescued by inhibition of the stringent response pathway or by overexpression of the editing domain of alanyl-tRNA^Ala synthetase that enables detoxification of tRNA misacylation. Furthermore, MurM can incorporate seryl groups from mischarged Seryl-tRNA^Ala_UGC into cell wall precursors with exquisite specificity. We conclude that MurM contributes to the fidelity of translation control and modulates the stress response by decreasing the pool of mischarged tRNAs. Finally, we show that enhanced lysis of ΔmurMN pneumococci is caused by LytA, and the murMN operon influences macrophage phagocytosis in a LytA-dependent manner. Thus, MurMN attenuates stress responses with consequences for host–pathogen interactions. Our data suggest a causal link between misaminoacylated tRNA accumulation and activation of the stringent response. In order to prevent potential corruption of translation, consumption of seryl-tRNA^Ala by MurM may represent a first line of defense. When this mechanism is overwhelmed or absent (ΔmurMN), the stringent response shuts down translation to avoid toxic generation of mistranslated/misfolded proteins.

Gram-positive bacteria have evolved a thick and sophisticated cell wall that ensures bacterial structural integrity and is critical for cellular viability. This dynamic structure includes peptidoglycan, surface anchored proteins, wall teichoic acids, lipoteichoic acids, lipoproteins, and capsular polysaccharides (1, 2). It is also a major target of immune defenses and antibiotics (3, 4). Bacteria with high pathogenic potential, such as Streptococcus pneumoniae (pneumococcus), encounter hostile environments within the human host, where the cell wall serves as a barrier and an interface between the bacterium and its host.The pneumococcal peptidoglycan consists of glycan chains that are cross-linked directly or indirectly via peptide bridges (5). These glycan chains consist of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) residues. Each NAM subunit is attached to a stem peptide which is cross-linked to an adjacent stem peptide from a nearby glycan chain. In the pneumococcus, this cross-link can be direct or indirect through a dipeptide branch that is assembled by MurM and MurN, two transfer RNA (tRNA)-dependent amino acyl transferases (6, 7). Indirect cross-linking requires the synthesis of lipid II-linked branched peptidoglycan precursors on the cytoplasmic face of the pneumococcal cell membrane. MurM transfers alanyl or seryl residues from aminoacyl-tRNAs to the ε-amino group of the stem peptide lysine of the peptidoglycan precursor lipid II (6), and MurN appends an alanyl residue from alanyl-tRNA^Ala to the residue added by MurM (7). Lipid precursors are then flipped to the extracellular face of the cell membrane to be polymerized by transglycosylation. The nascent glycan strands are then cross-linked through the third residue (l-Lys, which can be branched or unbranched) of a stem peptide emanating from one glycan strand to the fourth residue (D-Ala) of the stem peptide of an adjacent glycan strand by penicillin binding proteins (8, 9). Thus, pneumococcal peptidoglycan synthesis links stem peptides with a dipeptide comprising a C-terminal l-Ala or l-Ser and an N-terminal l-Ala.Allelic variants of murM lead to diversity in the nature of stem peptide branching in the cell wall peptidoglycan (10–12). Similarly, allelic variation among pneumococcal penicillin binding protein genes is responsible for drastically increasing the minimum inhibitory concentration for β-lactam antibiotics. However, such clinical high-level resistance to β-lactams additionally requires murMN as inactivation of this operon leads to a complete loss of penicillin resistance (8, 13).The employment of aminoacyl-tRNAs as MurM and MurN substrates juxtaposes the fundamental cellular functions of peptidoglycan biosynthesis and protein translation with each other and antibiotic resistance. The ability of MurM to incorporate both seryl and alanyl residues from tRNA into peptidoglycan branches is intriguing. Serine is an amino acid that is erroneously recognized by alanyl-tRNA^Ala synthetase (AlaRS), which itself possess editing mechanisms to forestall noncognate seryl-tRNA^Ala synthesis and consequent misincorporation of serine at alanine codons (14, 15).In vitro, MurM can deacylate seryl and alanyl-tRNA^Ala by hydrolysis (6, 16). This led to the hypothesis that MurM-catalyzed aminoacyl-tRNA deacylation provides an in trans mechanism for the correction of deficits in the editing activity and fidelity of AlaRS (16). However, several observations are inconsistent with the role of MurM in editing misacylation of tRNA via simple hydrolytic deacylation of a mischarged substrate: 1) initial reports were made with a vast excess of enzymes relative to the tRNA substrate which precluded demonstration of MurM catalysis (16), 2) MurM-supported aminoacyl-tRNA hydrolysis rates are negligible when compared with those of peptidoglycan precursor aminoacylation (6), and 3) MurM-mediated alanyl-tRNA^Ala deacylation is significantly more rapid than seryl-tRNA^Ala deacylation (16). Therefore, the tantalizing possibility that seryl-tRNA^Ala consumption by MurM-catalyzed lipid II serylation contributes to maintaining accuracy of alanine codon translation remains to be validated in the literature.Under stress, many bacterial cellular processes including translation are regulated by the stringent response pathway. Deprivation of amino acids or carbon sources, elevated temperature, and acidic conditions can trigger the stringent response and reconfigure cellular metabolism to adapt to challenging conditions to ensure bacterial survival (17–20). In addition, the stringent response plays a role in regulation of bacterial virulence and susceptibility to antimicrobials (18, 21–23). The stringent response pathway is characterized by the accumulation of guanosine tetra- (ppGpp) and pentaphosphate (pppGpp), collectively referred to as alarmones [(p)ppGpp]. In the majority of Gram-negative bacteria, these alarmones are produced by the synthetase RelA and hydrolyzed by SpoT. In these bacteria, binding of deacylated-tRNA to ribosomes activates alarmone production (24–27). In Gram-positive bacteria, these molecules are produced by a bifunctional RSH (RelA/SpoT homolog) protein, which possesses both alarmone synthetase and hydrolase activity (19, 28). Much less is understood about activation of the stringent response in Gram-positive bacteria. However, recent work in Bacillus subtilis suggests that alarmones target initiation factor 2 (IF2) and, in doing so, inhibit translation (29). Together, these data suggest a clear link between alarmones, tRNA targeting, and translation.In this study, we show that MurMN is a molecular link between cell wall biosynthesis and translation quality control through its preferential utilization of misaminoacylated tRNA for the formation of indirect cross-links in pneumococcal peptidoglycan. Our findings indicate that the absence of these proteins sensitizes pneumococcal cells to acidic stress. Additionally, we found that a murMN deletion strain presents growth defects when grown in mildly acidic conditions, similar to those in which the ability of AlaRS to edit serine misaminoacylation of tRNA^Ala is reduced. The impairment of AlaRS editing activity likely resulted in the accumulation of mischarged tRNA and the subsequent activation of the stringent response pathway. Furthermore, our data suggest that activation of the stringent response is associated with the modulation of LytA activity, and this is reflected in changes in the initiation of stationary phase-induced autolysis and in the extent of phagocytosis of pneumococci. These findings provide insight into cell wall function by suggesting that cell wall biosynthesis enzymes can buffer the deleterious consequences of intracellular stress on protein synthesis and modulate entry into the stringent response pathway.     

Characterization,stability, and application of domain walls in flexible mechanical metamaterials

Domain walls, commonly occurring at the interface of different phases in solid-state materials, have recently been harnessed at the structural scale to enable additional modes of functionality. Here, we combine experimental, numerical, and theoretical tools to investigate the domain walls emerging upon uniaxial compression in a mechanical metamaterial based on the rotating-squares mechanism. We first show that these interfaces can be generated and controlled by carefully arranging a few phase-inducing defects. We establish an analytical model to capture the evolution of the domain walls as a function of the applied deformation. We then employ this model as a guideline to realize interfaces of complex shape. Finally, we show that the engineered domain walls modify the global response of the metamaterial and can be effectively exploited to tune its stiffness as well as to guide the propagation of elastic waves.

The coexistence of two or more phases plays a central role in many ordered solid-state materials, including ferroelectrics (1–3), ferromagnets (4, 5), ferroelastics (6, 7), shape memory alloys (7, 8), and liquid crystals (9). Despite being intrinsically different, these materials all share the emergence of domain walls—a type of topological defect that separates regions of different phases (10). Such interfaces are crucial for the control of many material properties, including coercivity, resistance, and/or fatigue (11), and have also been exploited to enable logic operations (12), racetrack memory (13), and line scanners for reading optical memories (14). Inspired by the recent advancements in domain walls control strategies at the atomistic scale, researchers have designed a variety of nonlinear mechanical structures to support these interfaces (15–25). Domain walls engineered at the structural scale have facilitated the control of elastic pulses (16, 17, 19, 25), the encryption of information (23), and the realization of deployable structures (20) as well as of phase-transforming metamaterials (15, 18). However, due to the structural complexity of mechanical metamaterials, no analytical solution has been proposed that fully describes the physics of such domain walls. This limits their systematic application in the design of smart structures and devices and hinders the discovery of additional functionalities.Here, we use a combination of experiments and analyses to study the domain walls emerging in a mechanical metamaterial based on the rotating-squares mechanism. We start by introducing defects into the system to locally impose nucleation of one of the two supported buckling-induced rotated phases upon compression. Importantly, when such defects lead to the coexistence of two phases within the specimen, domain walls form, across which the angle of individual squares switches from one direction of rotation to the other. We establish an analytical model that fully describes the emerging domain walls, including their profile and position as a function of the applied deformation. Guided by our model, we then introduce pinning defects to reshape the energy landscape of the system and, therefore, engineer domain walls along arbitrary complex paths. Based on our findings we foresee the exploitation of domain walls in the realm of mechanical metamaterials to realize additional functionalities, as we hereby demonstrate by achieving stiffness tuning and reconfigurable elastic wave guiding.     

Circular swimming motility and disordered hyperuniform state in an algae system

Active matter comprises individually driven units that convert locally stored energy into mechanical motion. Interactions between driven units lead to a variety of nonequilibrium collective phenomena in active matter. One of such phenomena is anomalously large density fluctuations, which have been observed in both experiments and theories. Here we show that, on the contrary, density fluctuations in active matter can also be greatly suppressed. Our experiments are carried out with marine algae (Effrenium voratum), which swim in circles at the air–liquid interfaces with two different eukaryotic flagella. Cell swimming generates fluid flow that leads to effective repulsions between cells in the far field. The long-range nature of such repulsive interactions suppresses density fluctuations and generates disordered hyperuniform states under a wide range of density conditions. Emergence of hyperuniformity and associated scaling exponent are quantitatively reproduced in a numerical model whose main ingredients are effective hydrodynamic interactions and uncorrelated random cell motion. Our results demonstrate the existence of disordered hyperuniform states in active matter and suggest the possibility of using hydrodynamic flow for self-assembly in active matter.

Active matter exists over a wide range of spatial and temporal scales (1–6) from animal groups (7, 8) to robot swarms (9–11), to cell colonies and tissues (12–16), to cytoskeletal extracts (17–20), to man-made microswimmers (21–25). Constituent particles in active matter systems are driven out of thermal equilibrium at the individual level; they interact to develop a wealth of intriguing collective phenomena, including clustering (13, 22, 24), flocking (11, 26), swarming (12, 13), spontaneous flow (14, 20), and giant density fluctuations (10, 11). Many of these observed phenomena have been successfully described by particle-based or continuum models (1–6), which highlight the important roles of both individual motility and interparticle interactions in determining system dynamics.Current active matter research focuses primarily on linearly swimming particles which have a symmetric body and self-propel along one of the symmetry axes. However, a perfect alignment between the propulsion direction and body axis is rarely found in reality. Deviation from such a perfect alignment leads to a persistent curvature in the microswimmer trajectories; examples of such circle microswimmers include anisotropic artificial micromotors (27, 28), self-propelled nematic droplets (29, 30), magnetotactic bacteria and Janus particles in rotating external fields (31, 32), Janus particle in viscoelastic medium (33), and sperm and bacteria near interfaces (34, 35). Chiral motility of circle microswimmers, as predicted by theoretical and numerical investigations, can lead to a range of interesting collective phenomena in circular microswimmers, including vortex structures (36, 37), localization in traps (38), enhanced flocking (39), and hyperuniform states (40). However, experimental verifications of these predictions are limited (32, 35), a situation mainly due to the scarcity of suitable experimental systems.Here we address this challenge by investigating marine algae Effrenium voratum (41, 42). At air–liquid interfaces, E. voratum cells swim in circles via two eukaryotic flagella: a transverse flagellum encircling the cellular anteroposterior axis and a longitudinal one running posteriorly. Over a wide range of densities, circling E. voratum cells self-organize into disordered hyperuniform states with suppressed density fluctuations at large length scales. Hyperuniformity (43, 44) has been considered as a new form of material order which leads to novel functionalities (45–49); it has been observed in many systems, including avian photoreceptor patterns (50), amorphous ices (51), amorphous silica (52), ultracold atoms (53), soft matter systems (54–61), and stochastic models (62–64). Our work demonstrates the existence of hyperuniformity in active matter and shows that hydrodynamic interactions can be used to construct hyperuniform states.     

A catalog of tens of thousands of viruses from human metagenomes reveals hidden associations with chronic diseases

Despite remarkable strides in microbiome research, the viral component of the microbiome has generally presented a more challenging target than the bacteriome. This gap persists, even though many thousands of shotgun sequencing runs from human metagenomic samples exist in public databases, and all of them encompass large amounts of viral sequence data. The lack of a comprehensive database for human-associated viruses has historically stymied efforts to interrogate the impact of the virome on human health. This study probes thousands of datasets to uncover sequences from over 45,000 unique virus taxa, with historically high per-genome completeness. Large publicly available case-control studies are reanalyzed, and over 2,200 strong virus–disease associations are found.

The human virome is the sum total of all viruses that are intimately associated with people. This includes viruses that directly infect human cells (1, 2) but mostly consists of viruses infecting resident bacteria (i.e., phages) (3). While the large majority of microbiome studies have focused on the bacteriome, revealing numerous important functions for bacteria in human physiology (4), information about the human virome has lagged. However, a number of recent studies have begun making inroads into characterizing the virome (5–13).Just as human-tropic viruses can have dramatic effects on people, phages are able to dramatically alter bacterial physiology and regulate host population size. A variety of evolutionary dynamics can be at play in the phage/bacterium arena, including Red Queen (11), arms-race (14), and piggyback-the-winner (15) relationships, to name just a few. In the gut, many phages enter a lysogenic or latent state and are retained as integrated or episomal prophages within the host bacterium (16). In some instances, the prophage can buttress host fitness (at least temporarily) rather than destroy the host cell. To this effect, prophages often encode genes that can dramatically alter the phenotype of the bacteria, such as toxins (17), virulence factors (18), antibiotic resistance genes (19), photosystem components (20), other auxiliary metabolic genes (21), and CRISPR-Cas systems (22), along with countless genes of unknown function. Experimental evidence has shown that bacteria infected with particular phages (i.e., “virocells”) are physiologically distinct from cognate bacteria that lack those particular phages (21).There have been a few documented cases in which phages have been shown to be mechanistically involved in human health and disease, sometimes through direct interactions with human cells. This includes roles in increased bacterial virulence (17), response to cancer immunotherapy (23), clearance of bacterial infection (24), and resistance to antibiotics (25). Furthermore, phage therapy, the targeted killing of specific bacteria using live phage particles, has shown increasing promise for treatment of antibiotic-resistant bacterial infections (26). Considering the progress already made, phages represent attractive targets of and tools for microbiome restructuring in the interest of improving health outcomes.In addition, several studies have conducted massively parallel sequencing on virus-enriched samples of human stool, finding differential abundance of some phages in disease conditions (6, 27–29). A major issue encountered by these studies is that there is not yet a comprehensive database of annotated virus genome sequences, and de novo prediction of virus sequences from metagenomic assemblies remains a daunting challenge (3). Further, though some tools are able to predict virus-derived sequences with high specificity (30, 31), these tools have not been applied to human metagenomes at a large scale [with a possible exception (13)], and, regrettably, most uncovered virus genomes do not end up in central repositories. One study suggests that only 31% of the assembled sequence data in virion-enriched virome surveys could be identified as recognizably viral (32). On the other hand, another study of 12 individuals was able to recruit over 80% of reads from virus-enriched samples to putative virus contigs (11). Still, most of the potential viral contigs from this study were unclassifiable sequences, and a large majority of contigs appeared to represent subgenomic fragments under 10 kb.The current study sought to overcome the traditional challenges of sparse viral databases and poor detection of highly divergent viral sequences by using Cenote-Taker 2, a new virus discovery and annotation tool (33). The pipeline was applied to sequencing data from nearly 6,000 human metagenome samples. Strict criteria identified over 180,000 viral contigs representing 45,033 specific taxa. In most cases, 70 to 99% of reads from virus-enriched stool datasets could be back-aligned to the Cenote-Taker 2–compiled Human Virome Database. Furthermore, the curated database allowed read-alignment–based abundance profiling of the virome in human metagenomic datasets, enabling the reanalysis of a panel of existing case-control studies. The reanalysis revealed previously undetected associations between chronic diseases and the abundance of 2,265 specific virus taxa.     

Characterizing the “fungal shunt”: Parasitic fungi on diatoms affect carbon flow and bacterial communities in aquatic microbial food webs

Microbial interactions in aquatic environments profoundly affect global biogeochemical cycles, but the role of microparasites has been largely overlooked. Using a model pathosystem, we studied hitherto cryptic interactions between microparasitic fungi (chytrid Rhizophydiales), their diatom host Asterionella, and cell-associated and free-living bacteria. We analyzed the effect of fungal infections on microbial abundances, bacterial taxonomy, cell-to-cell carbon transfer, and cell-specific nitrate-based growth using microscopy (e.g., fluorescence in situ hybridization), 16S rRNA gene amplicon sequencing, and secondary ion mass spectrometry. Bacterial abundances were 2 to 4 times higher on individual fungal-infected diatoms compared to healthy diatoms, particularly involving Burkholderiales. Furthermore, taxonomic compositions of both diatom-associated and free-living bacteria were significantly different between noninfected and fungal-infected cocultures. The fungal microparasite, including diatom-associated sporangia and free-swimming zoospores, derived ∼100% of their carbon content from the diatom. By comparison, transfer efficiencies of photosynthetic carbon were lower to diatom-associated bacteria (67 to 98%), with a high cell-to-cell variability, and even lower to free-living bacteria (32%). Likewise, nitrate-based growth for the diatom and fungi was synchronized and faster than for diatom-associated and free-living bacteria. In a natural lacustrine system, where infection prevalence reached 54%, we calculated that 20% of the total diatom-derived photosynthetic carbon was shunted to the parasitic fungi, which can be grazed by zooplankton, thereby accelerating carbon transfer to higher trophic levels and bypassing the microbial loop. The herein termed “fungal shunt” can thus significantly modify the fate of photosynthetic carbon and the nature of phytoplankton–bacteria interactions, with implications for diverse pelagic food webs and global biogeochemical cycles.

Parasitism is one of the most common consumer strategies on Earth (1–3). Recently, it has also been identified as one of the dominating interactions within the planktonic interactome (4, 5), and yet parasites remain poorly considered in analyses of trophic interactions and element cycling in aquatic systems (6, 7). The foundation of trophic interactions in plankton communities is set by single-cell phytoplankton, which contributes almost half of the world’s primary production (8). According to our common understanding, the newly fixed carbon (C) is channeled either through the microbial loop, classical food web, or viral shunt, which supports the growth of heterotrophic bacteria and nanoflagellates, zooplankton and higher trophic levels, or viruses, respectively (9). However, fungi, particularly fungal microparasites, are rarely considered as contributors to C and nutrient cycling, although they are present and active in diverse aquatic environments (10–12).Members of the fungal division Chytridiomycota, referred to as chytrids, can thrive as microparasites on phytoplankton cells in freshwater (11, 13, 14) and marine systems (15–17), infecting up to 90% of the phytoplankton host population (18–21). A recent concept, called mycoloop, describes parasitic chytrids as an integral part of aquatic food webs (22). Energy and organic matter are thereby transferred from large, often inedible phytoplankton to chytrid zoospores, which are consumed by zooplankton (23–27). Hence, parasitic chytrids establish a novel trophic link between phytoplankton and zooplankton. Our understanding of element cycling and microbial interactions during chytrid epidemics, however, remains sparse. For instance, the cell-to-cell C transfer from single phytoplankton cells to their directly associated chytrids has not been quantified to date. Moreover, the relationship between parasitic chytrids and heterotrophic bacteria is largely undescribed.Phytoplankton cells release substantial amounts of dissolved organic C (DOC) (28), whereby up to 50% of photosynthetic C is consumed as DOC by bacteria (29–32). Thus, bacterial communities are intimately linked to phytoplankton abundances and production (33). Phytoplankton–bacteria interactions are particularly strong within the phycosphere, the region immediately surrounding individual phytoplankton cells (33–35), where nutrient concentrations are several-folds higher compared to the ambient water (36, 37), and nutrient assimilation rates of phytoplankton-associated bacteria are at least twice as fast as those of their free-living counterparts (38, 39). Importantly, parasitic chytrids may distort these phytoplankton–bacteria interactions within and outside the phycosphere since they modulate substrate and nutrient availabilities and presumably also bacterial activity and community composition. The effects of this distortion are virtually unresolved, but the few available data indicate that chytrid infections alter the composition and concentration of DOC (40), while abundances of free-living bacteria increase (25, 40) or remain unchanged (24).To disentangle phytoplankton–fungi–bacteria interactions at a microspatial single-cell scale—the scale at which phytoplankton, fungi, and bacteria intimately interact—we used one of the few existing model pathosystems, composed of the freshwater diatom Asterionella formosa, the chytrid Rhizophydiales sp., and coenriched populations of heterotrophic bacteria. Our methodology included dual stable-isotope incubations (¹³C-bicarbonate and ¹⁵N-nitrate), single-cell–resolution secondary ion mass spectrometry (SIMS) (IMS 1280 and NanoSIMS 50L), 16S rRNA gene/16S rRNA sequencing, microscopy (e.g., fluorescence in situ hybridization [FISH]), and nutrient analyses. We particularly focused on the initial C transfer from the phytoplankton host to parasitic chytrids, which we term the “fungal shunt,” as part of the mycoloop. The objectives were twofold: 1) quantifying the transfer of photosynthetic C from phytoplankton cells to infectious chytrids, cell-associated bacteria, and free-living bacteria and 2) characterizing the effect of parasitic fungi on bacterial populations, considering bacterial abundances, bacterial–diatom attachment, single-cell activity rates, and community composition. The obtained data challenge the common perception of aquatic microbial food webs by demonstrating the significant role that parasitic fungi can play in microbial community structure, interactions, and element cycling during phytoplankton growth.     

A multiplier peroxiporin signal transduction pathway powers piscine spermatozoa

The primary task of a spermatozoon is to deliver its nuclear payload to the egg to form the next-generation zygote. With polyandry repeatedly evolving in the animal kingdom, however, sperm competition has become widespread, with the highest known intensities occurring in fish. Yet, the molecular controls regulating spermatozoon swimming performance in these organisms are largely unknown. Here, we show that the kinematic properties of postactivated piscine spermatozoa are regulated through a conserved trafficking mechanism whereby a peroxiporin ortholog of mammalian aquaporin-8 (Aqp8bb) is inserted into the inner mitochondrial membrane to facilitate H₂O₂ efflux in order to maintain ATP production. In teleosts from more ancestral lineages, such as the zebrafish (Danio rerio) and the Atlantic salmon (Salmo salar), in which spermatozoa are activated in freshwater, an intracellular Ca²⁺-signaling directly regulates this mechanism through monophosphorylation of the Aqp8bb N terminus. In contrast, in more recently evolved marine teleosts, such the gilthead seabream (Sparus aurata), in which spermatozoa activation occurs in seawater, a cross-talk between Ca²⁺- and oxidative stress-activated pathways generate a multiplier regulation of channel trafficking via dual N-terminal phosphorylation. These findings reveal that teleost spermatozoa evolved increasingly sophisticated detoxification pathways to maintain swimming performance under a high osmotic stress, and provide insight into molecular traits that are advantageous for postcopulatory sexual selection.

For many dioecious animals, spermatozoon velocity, progressivity, and duration of motility are vital determinants of reproductive success and are thus major selection criteria for sperm evolution (1–6). Maximizing such kinematic properties contributes to spermatozoon vigor (7); however, due to the limitations in sperm ATP stores, which provide the chemical energy for flagellar contractions, a trade-off between swimming fast and for extended periods typically exists (8). Optimal combinations of traits that improve spermatozoon vigor are nevertheless important in polyandrous vertebrates facing sperm competition, which represents a powerful form of postcopulatory sexual selection (9–15). Since the phenomenon of sperm competition was first recognized (16), investigators have sought to understand the underlying mechanisms that could explain advantageous trait selection (17). To date, however, most research has focused on the physical and morphological properties involved in sperm competition, and very little is known concerning the molecular and genetic mechanisms underpinning spermatozoon performance (15, 18, 19).One positively selected morphological change in respect of spermatozoon velocity and longevity in vertebrates as diverse as fishes, birds, and mammals, has been the increase in the spermatozoon midpiece size and the number or scale of mitochondria therein (20–22). Such changes have logically been associated with increased mitochondrial production of ATP for improved flagellar motility. However, the biochemical reactions that lead to increased ATP synthesis also generate elevated levels of hydrogen peroxide (H₂O₂), a reactive oxygen species (ROS) that inhibits mitochondrial function and suppresses flagellar motility (23–27). With osmotic stress of the exposed ejaculate generating additional ROS (26, 28), it has been unclear how sperm evolved molecular mechanisms that surmount such signaling conflicts.A solution to this apparent paradox was recently discovered in the spermatozoa of a marine teleost, in which a water channel protein, now termed Aqp8bb (a peroxiporin ortholog of mammalian aquaporin-8) (29), is rapidly (<1 s) trafficked to the inner mitochondrial membrane upon activation in seawater (SW) to facilitate H₂O₂ efflux and the maintenance of ATP production and flagellar motility (26). The importance of Aqp8bb, which mainly functions as a peroxiporin in these germ cells, was demonstrated through immunological inhibitory experiments, which highlighted the channel-trafficking mechanism as a critical regulator of the spermatozoon velocity and motility (26). To date, however, the signal transduction pathways that regulate peroxiporin trafficking in vertebrate spermatozoa remain completely unknown.Among externally fertilizing vertebrates, the highest known intensity of sperm competition occurs in true bony fishes (teleosts) (6), and we therefore focused our investigations on these model organims. In contrast to amniotic vertebrates, in which ejaculates become gelatinous when emitted (30), the ejaculates of freshwater (FW) and marine teleosts are not only rapidly diluted, but respectively face tremendous and opposing osmotic stresses, which in most species activate sperm motility (31–33). To understand the significance of such harsh environments for peroxiporin signal transduction pathway evolution, we selected model species from ancient and modern lineages of teleosts, including the FW ostariphysan zebrafish (Danio rerio), the FW protacanthopterygian Atlantic salmon (Salmo salar), and the modern marine acanthomorph gilthead seabream (Sparus aurata). Using a combination of pharmacological, molecular, and physiological approaches, we uncover the evolution of increasingly sophisticated peroxiporin signal transduction pathways powering their spermatozoa. The findings provide insight into the underlying hierarchy of systemic molecular traits that regulate the velocity, progressivity and duration of spermatozoon motility.     

The cyanobacterial taxis protein HmpF regulates type IV pilus activity in response to light

Motility is ubiquitous in prokaryotic organisms including the photosynthetic cyanobacteria where surface motility powered by type 4 pili (T4P) is common and facilitates phototaxis to seek out favorable light environments. In cyanobacteria, chemotaxis-like systems are known to regulate motility and phototaxis. The characterized phototaxis systems rely on methyl-accepting chemotaxis proteins containing bilin-binding GAF domains capable of directly sensing light, and the mechanism by which they regulate the T4P is largely undefined. In this study we demonstrate that cyanobacteria possess a second, GAF-independent, means of sensing light to regulate motility and provide insight into how a chemotaxis-like system regulates the T4P motors. A combination of genetic, cytological, and protein–protein interaction analyses, along with experiments using the proton ionophore carbonyl cyanide m-chlorophenyl hydrazine, indicate that the Hmp chemotaxis-like system of the model filamentous cyanobacterium Nostoc punctiforme is capable of sensing light indirectly, possibly via alterations in proton motive force, and modulates direct interaction between the cyanobacterial taxis protein HmpF, and Hfq, PilT1, and PilT2 to regulate the T4P motors. Given that the Hmp system is widely conserved in cyanobacteria, and the finding from this study that orthologs of HmpF and T4P proteins from the distantly related model unicellular cyanobacterium Synechocystis sp. strain PCC6803 interact in a similar manner to their N. punctiforme counterparts, it is likely that this represents a ubiquitous means of regulating motility in response to light in cyanobacteria.

Motility is ubiquitous in prokaryotic organisms, including both swimming motility in aqueous environments and twitching or gliding motility on solid surfaces, and enables these organisms to optimize their position in response to various environmental factors. Among the photosynthetic cyanobacteria, surface motility is widespread and facilitates phototaxis to seek out favorable light environments (1, 2), and, for multicellular filamentous cyanobacteria, plays a key role in dispersal as well as the establishment of nitrogen-fixing symbioses with eukaryotes (3) and the formation of supracellular structures (3–5).Current understanding of cyanobacterial surface motility at the molecular level has been informed primarily by studies of two model organisms, the unicellular strain Synechocystis sp. strain PCC6803 (herein Synechocystis) and the filamentous strain Nostoc punctiforme ATCC29133/{"type":"entrez-protein","attrs":{"text":"PCC73102","term_id":"1245706357"}}PCC73102, where motility is exhibited only by differentiated filaments termed “hormogonia.” Motility in both organisms is powered by a type IV pilus (T4P) system where the ATPases PilB and PilT drive the extension and subsequent retraction, respectively, of pili which adhere to the substrate and pull the cells forward (for review, see ref. 6). In Synechocystis, the T4P motors are distributed throughout the entire cell, allowing a 360 ° range of motion (7), whereas in N. punctiforme they are confined to rings at the cell poles (8), resulting in movement only along the long axis of the filament. Comparative genomics implies that this mechanism of motility is widely conserved among cyanobacteria (9).Both Synechocystis and N. punctiforme employ chemotaxis-like systems to regulate motility. One of these systems, the Hmp chemotaxis-like system of N. punctiforme (3, 10), and its orthologous counterpart, the Pil chemotaxis-like system of Synechocystis (11), includes homologs to the canonical Escherichia coli chemotaxis complex (for review, see ref. 12), including the histidine kinase CheA, the adaptor protein CheW, the response regulator CheY, and the methyl-accepting chemotaxis protein MCP. These systems are essential for motility in their respective organisms and appear to regulate the T4P motors, although there are distinct differences in the phenotypes for inactivation of the components from each. In Synechocystis, null mutations either enhance or reduce the level of surface piliation (11), whereas in N. punctiforme they disrupt the coordinated polarity, but not the overall level of piliation, and affect various other aspects of hormogonium development (3, 10). In N. punctiforme, the subcellular localization of this system has been determined and has been found arrayed in static, bipolar rings similar to the T4P motors (3). However, the signals that are perceived by the MCPs and the precise mechanism by which these systems modulate T4P activity is currently undefined.Recently, an additional component of the Hmp system, HmpF, was characterized (9). HmpF is a predicted coiled-coil protein and is ubiquitous to, but confined within, the cyanobacterial lineage (9). It is essential for accumulation of surface pili and exhibits dynamic, unipolar localization to the leading poles of most cells in hormogonium filaments (9). Based on these findings, a model has been proposed where the localization of HmpF is regulated by the other components of the Hmp system, and in turn, the unipolar accumulation of HmpF leads to the activation of the T4P motors on one side of the cell to facilitate directional movement.A second chemotaxis-like system in each organism, the Ptx system of N. punctiforme (13) and the Pix system of Synechocystis (14, 15), is essential for positive phototaxis. These systems contain MCPs with cyanobacteriochrome sensory domains capable of perceiving light (for review, see ref. 16). Disruption of the Pix system results in negative phototaxis under light conditions that normally produce a positive phototactic response (14). Several other proteins containing cyanobacteriochromes, and one containing a BLUF domain, also modulate phototaxis in Synechocystis (for review, see ref. 6). In N. punctiforme, disruption of the Ptx system abolishes the phototactic response completely, resulting in uniform movement in all directions regardless of the light conditions (13), and there are currently no other proteins reported to modulate phototaxis. More recently, a motile, wild isolate of the model unicellular cyanobacterium Synechococcus elongatus sp. PCC7942 was shown to possess a chemotaxis-like system that modulates phototaxis in a manner similar to that of the N. punctiforme Ptx system (17). How these systems influence T4P activity to facilitate phototaxis is also currently unknown.There is also a substantial body of literature on motility and phototaxis in cyanobacteria, primarily based on observational studies of various filamentous strains, that predates the development of genetically tractable model organisms (for review, see ref. 18). These reports suggested that the photosystems may serve a sensory role in modulating phototaxis and that proton motive force (PMF) powers motility (19, 20), a finding that is inconsistent with the theory that cyanobacteria possess a common T4P-based gliding motor driven by ATP hydrolysis. In this study, we help reconcile this historical data with more recent molecular studies by providing evidence that the Hmp chemotaxis-like system senses light, possibly indirectly through alterations in PMF, and in turn modulates the interaction of HmpF with the T4P base to activate the motors.     

Macrophage LC3-associated phagocytosis is an immune defense against Streptococcus pneumoniae that diminishes with host aging

Streptococcus pneumoniae is a leading cause of pneumonia and invasive disease, particularly, in the elderly. S. pneumoniae lung infection of aged mice is associated with high bacterial burdens and detrimental inflammatory responses. Macrophages can clear microorganisms and modulate inflammation through two distinct lysosomal trafficking pathways that involve 1A/1B-light chain 3 (LC3)-marked organelles, canonical autophagy, and LC3-associated phagocytosis (LAP). The S. pneumoniae pore-forming toxin pneumolysin (PLY) triggers an autophagic response in nonphagocytic cells, but the role of LAP in macrophage defense against S. pneumoniae or in age-related susceptibility to infection is unexplored. We found that infection of murine bone-marrow-derived macrophages (BMDMs) by PLY-producing S. pneumoniae triggered Atg5- and Atg7-dependent recruitment of LC3 to S. pneumoniae-containing vesicles. The association of LC3 with S. pneumoniae-containing phagosomes required components specific for LAP, such as Rubicon and the NADPH oxidase, but not factors, such as Ulk1, FIP200, or Atg14, required specifically for canonical autophagy. In addition, S. pneumoniae was sequestered within single-membrane compartments indicative of LAP. Importantly, compared to BMDMs from young (2-mo-old) mice, BMDMs from aged (20- to 22-mo-old) mice infected with S. pneumoniae were not only deficient in LAP and bacterial killing, but also produced higher levels of proinflammatory cytokines. Inhibition of LAP enhanced S. pneumoniae survival and cytokine responses in BMDMs from young but not aged mice. Thus, LAP is an important innate immune defense employed by BMDMs to control S. pneumoniae infection and concomitant inflammation, one that diminishes with age and may contribute to age-related susceptibility to this important pathogen.

Streptococcus pneumoniae (pneumococcus) commonly colonizes the nasopharynx asymptomatically but is also capable of infecting the lower respiratory tract to cause pneumonia and spreading to the bloodstream to cause septicemia and meningitis (1). Susceptibility to pneumonia and invasive disease caused by S. pneumoniae is remarkably higher in individuals aged 65 and over, leading to high rates of mortality and morbidity in the elderly population (1, 2). In countries, such as the United States and Japan, deaths due to pneumococcal pneumonia have been on the rise in parallel with the rapid growth in the elderly population (3, 4).A hallmark of pneumococcal pneumonia is a rapid and exuberant response by immune cells, such as neutrophils and macrophages. This innate immune response to S. pneumoniae lung infection is critical for pathogen clearance and the control of disease (5–7). Deficiencies in the number or function of innate phagocytic cells, such as neutropenia (8) or macrophage phagocytic receptor defects (9–12), lead to diminished pneumococcal clearance and increased risk of invasive pneumococcal disease in both mouse models and humans. Phagocytic activity in alveolar macrophages is important during early responses to subclinical infections (13–15), and during moderate S. pneumoniae lung infection, newly generated monocytes eggress from the bone marrow and migrate into the lungs, differentiating into monocyte-derived alveolar macrophages (16). In addition to directly eliminating the invading microbe, macrophages secrete key cytokines, such as tumor necrosis factor (TNF), interleukin-1β (IL-1β), and interleukin-6 (IL-6), that regulate effector cell functions and pulmonary inflammation (17–19).Although an innate immune response is critical for pathogen clearance, poorly controlled inflammation can lead to tissue damage and mortality (20, 21). For example, in murine models, neutrophilic infiltration can enhance pulmonary damage and disrupt epithelial barrier function, leading to bacteremia and mortality (22–25). Macrophages are critical not only in regulating the early inflammatory response, but are also crucial for curtailing inflammation during the resolution phase of infection to limit tissue damage and promote healing (26, 27).Elderly individuals have higher baseline and induced levels of inflammation, a phenomenon termed inflammaging (28), that contributes to many age-associated pathological conditions, including increased susceptibility to a variety of infectious diseases, such as S. pneumoniae infection (7, 28–30). S. pneumoniae-induced inflammation, characterized by increased levels of chemokines, proinflammatory cytokines, and decreased anti-inflammatory cytokines, such as IL-10, is enhanced in the elderly (29, 31) as well as in aged mice (32, 33) and correlates with ineffective immune responses. Age-related chronic exposure to TNF-α, for instance, dampens macrophage-mediated S. pneumoniae clearance during lung infection (34), and NLR family pyrin domain containing 3 inflammasome activation in macrophages diminishes upon aging in mice (35). However, the age-related changes in macrophage effector functions leading to diminished clearance of S. pneumoniae are incompletely understood.One important means of macrophage-mediated pathogen clearance is1A/1B-light chain-3 (LC3)-associated phagocytosis (LAP), a process by which cells target phagocytosed extracellular particles for efficient degradation (36–38). LAP combines the molecular machineries of phagocytosis and autophagy, resulting in the conjugation of the autophagic marker, the microtubule-associated protein LC3, to phosphatidylethanolamine on the phagosomal membrane, generating so-called “LAPosomes” that undergo facilitated fusion with lysosomes (38, 39). Canonical autophagy targets cytoplasmic components, such as damaged subcellular organelles and intracellular microbes for sequestration into double-membrane autophagic vesicles (40, 41). In contrast, LAPosomes retain the single-membrane nature of phagosomes, and their formation requires overlapping but nonidentical genes compared to canonical autophagy (42). In addition to enabling efficient degradation of phagocytosed bacteria, LAP also plays important immune regulatory roles, such as in curtailing proinflammatory cytokine production during the subsequent innate immune response (39, 43). Indeed, the LAP-mediated microbial defense and immunomodulatory functions work together to limit tissue damage and restore homeostasis (38).S. pneumoniae triggers canonical autophagy in epithelial cells and fibroblasts, and bacteria can be found in double-membrane vacuoles whose formation is dependent on autophagic machinery (44). Many bacterial pathogens that induce autophagy produce pore-forming toxins, which can damage endosomal membranes, thus, recruiting autophagic machinery to engulf injured organelles (45). Pneumococcus-induced autophagy is dependent on the cholesterol-dependent pore-forming toxin pneumolysin (PLY), which triggers the autophagic delivery of S. pneumoniae to lysosomes and results in bacterial killing (44, 46). Recently, a kinetic examination of S. pneumoniae-targeting autophagy in fibroblasts demonstrated that canonical autophagy was preceded by early and rapid PLY-dependent LAP (47). However, the requirements for this process were somewhat different from LAP in macrophages, and the pneumococcus-containing LAPosomes did not promote bacterial clearance but required subsequent transition to canonical autophagy to reduce bacterial numbers (46, 47).In the current study, we found that S. pneumoniae infection of murine bone-marrow-derived macrophages (BMDMs) induces LAP in a PLY-dependent manners and that age-related defects in BMDM LAP contributed to diminished bactericidal activity and enhanced proinflammatory cytokine production. Our results suggest that PLY-induced LAP promotes bacterial clearance, and age-associated dysregulation of this process may contribute to enhanced bacterial survival, poorly regulated inflammation, and increased susceptibility to invasive pneumococcal disease.     

Defining principles that influence antimicrobial peptide activity against capsulated Klebsiella pneumoniae

The extracellular polysaccharide capsule of Klebsiella pneumoniae resists penetration by antimicrobials and protects the bacteria from the innate immune system. Host antimicrobial peptides are inactivated by the capsule as it impedes their penetration to the bacterial membrane. While the capsule sequesters most peptides, a few antimicrobial peptides have been identified that retain activity against encapsulated K. pneumoniae, suggesting that this bacterial defense can be overcome. However, it is unclear what factors allow peptides to avoid capsule inhibition. To address this, we created a peptide analog with strong antimicrobial activity toward several K. pneumoniae strains from a previously inactive peptide. We characterized the effects of these two peptides on K. pneumoniae, along with their physical interactions with K. pneumoniae capsule. Both peptides disrupted bacterial cell membranes, but only the active peptide displayed this activity against capsulated K. pneumoniae. Unexpectedly, the active peptide showed no decrease in capsule binding, but did lose secondary structure in a capsule-dependent fashion compared with the inactive parent peptide. We found that these characteristics are associated with capsule-peptide aggregation, leading to disruption of the K. pneumoniae capsule. Our findings reveal a potential mechanism for disrupting the protective barrier that K. pneumoniae uses to avoid the immune system and last-resort antibiotics.

Multidrug-resistant (MDR) bacterial infections have become a major threat to human health (1–3). Mortality rates from infections caused by gram-negative bacteria, specifically Klebsiella pneumoniae, are on the rise owing to the lack of effective antibiotics to treat the emergent MDR strains (4–7). The capsule of K. pneumoniae is composed of extracellular polysaccharides that promote infection by masking the bacteria from immune recognition and provide an especially potent barrier against peptide-based antimicrobials, including innate host defense peptides and last-resort polymyxin antibiotics (8–14).Antimicrobial peptides are commonly amphipathic, with both a charged and a hydrophobic character (15). The anionic nature of the bacterial capsule promotes an electrostatic attraction to cationic antimicrobial peptides, and peptide hydrophobicity has been proposed to enhance capsule binding through nonionic interactions (9, 12, 16). Interaction with the bacterial capsule is thought to induce structural changes that cause sequestration of antimicrobial peptides to prevent them from reaching their bacterial membrane target (16, 17). While the bacterial capsule inhibits host defense peptides and polymyxins, a few amphipathic antimicrobial peptides have been identified that can retain activity against capsulated K. pneumoniae (18–21). However, it is not known what enables some peptides to avoid sequestration by the capsule of K. pneumoniae while the capsule effectively neutralizes our innate host defense peptides with similar physicochemical properties. This lack of knowledge prevents us from understanding how to bypass the capsule barrier that K. pneumoniae uses to avoid our innate immune response and last-resort treatment options.Here we characterize the synthetic evolution of a peptide inhibited by capsule to a peptide with potent activity against capsulated K. pneumoniae. Remarkably, our results indicate that rather than reduced interactions, our active peptide retains binding to capsule and undergoes conformational changes associated with capsule aggregation. We present a model in which peptide-driven sequestration of capsule disrupts this barrier and reduces its ability to protect K. pneumoniae against antimicrobial attack. These findings provide insight into improving antimicrobial peptide activity against K. pneumoniae and may help strengthen our understanding of the inability of innate host defense peptides to act on capsulated bacteria.     

From the Cover: Evidence from South Africa for a protracted end-Permian extinction on land

Earth’s largest biotic crisis occurred during the Permo–Triassic Transition (PTT). On land, this event witnessed a turnover from synapsid- to archosauromorph-dominated assemblages and a restructuring of terrestrial ecosystems. However, understanding extinction patterns has been limited by a lack of high-precision fossil occurrence data to resolve events on submillion-year timescales. We analyzed a unique database of 588 fossil tetrapod specimens from South Africa’s Karoo Basin, spanning ∼4 My, and 13 stratigraphic bin intervals averaging 300,000 y each. Using sample-standardized methods, we characterized faunal assemblage dynamics during the PTT. High regional extinction rates occurred through a protracted interval of ∼1 Ma, initially co-occurring with low origination rates. This resulted in declining diversity up to the acme of extinction near the Daptocephalus–Lystrosaurus declivis Assemblage Zone boundary. Regional origination rates increased abruptly above this boundary, co-occurring with high extinction rates to drive rapid turnover and an assemblage of short-lived species symptomatic of ecosystem instability. The “disaster taxon” Lystrosaurus shows a long-term trend of increasing abundance initiated in the latest Permian. Lystrosaurus comprised 54% of all specimens by the onset of mass extinction and 70% in the extinction aftermath. This early Lystrosaurus abundance suggests its expansion was facilitated by environmental changes rather than by ecological opportunity following the extinctions of other species as commonly assumed for disaster taxa. Our findings conservatively place the Karoo extinction interval closer in time, but not coeval with, the more rapid marine event and reveal key differences between the PTT extinctions on land and in the oceans.

Mass extinctions are major perturbations of the biosphere resulting from a wide range of different causes including glaciations and sea level fall (1), large igneous provinces (2), and bolide impacts (3, 4). These events caused permanent changes to Earth’s ecosystems, altering the evolutionary trajectory of life (5). However, links between the broad causal factors of mass extinctions and the biological and ecological disturbances that lead to species extinctions have been difficult to characterize. This is because ecological disturbances unfold on timescales much shorter than the typical resolution of paleontological studies (6), particularly in the terrestrial record (6–8). Coarse-resolution studies have demonstrated key mass extinction phenomena including high extinction rates and lineage turnover (7, 9), changes in species richness (10), ecosystem instability (11), and the occurrence of disaster taxa (12). However, finer time resolutions are central to determining the association and relative timings of these effects, their potential causal factors, and their interrelationships. Achieving these goals represents a key advance in understanding the ecological mechanisms of mass extinctions.The end-Permian mass extinction (ca. 251.9 Ma) was Earth’s largest biotic crisis as measured by taxon last occurrences (13–15). Large outpourings from Siberian Trap volcanism (2) are the likely trigger of calamitous climatic changes, including a runaway greenhouse effect and ocean acidification, which had profound consequences for life on land and in the oceans (16–18). An estimated 81% of marine species (19) and 89% of tetrapod genera became extinct as established Permian ecosystems gave way to those of the Triassic. In the ocean, this included the complete extinction of reef-forming tabulate and rugose corals (20, 21) and significant losses in previously diverse ammonoid, brachiopod, and crinoid families (22). On land, many nonmammalian synapsids became extinct (16), and the glossopterid-dominated floras of Gondwana also disappeared (23). Stratigraphic sequences document a global “coral gap” and “coal gap” (24, 25), suggesting reef and forest ecosystems were rare or absent for up to 5 My after the event (26). Continuous fossil-bearing deposits documenting patterns of turnover across the Permian–Triassic transition (PTT) on land (27) and in the oceans (28) are geographically widespread (29, 30), including marine and continental successions that are known from China (31, 32) and India (33). Continental successions are known from Russia (34), Australia (35), Antarctica (36), and South Africa’s Karoo Basin (Fig. 1 and 37–40), the latter providing arguably the most densely sampled and taxonomically scrutinized (41–43) continental record of the PTT. The main extinction has been proposed to occur at the boundary between two biostratigraphic zones with distinctive faunal assemblages, the Daptocephalus and Lystrosaurus declivis assemblage zones (Fig. 1), which marks the traditional placement of the Permian–Triassic geologic boundary [(37) but see ref. 44]. Considerable research has attempted to understand the anatomy of the PTT in South Africa (38, 39, 45–52) and to place it in the context of biodiversity changes across southern Gondwana (53, 54) and globally (29, 31, 32, 44, 47, 55).Open in a separate windowFig. 1.Map of South Africa depicting the distribution of the four tetrapod fossil assemblage zones (Cistecephalus, Daptocephalus, Lystrosaurus declivis, Cynognathus) and our two study sites where fossils were collected in this study (sites A and B). Regional lithostratigraphy and biostratigraphy within the study interval are shown alongside isotope dilution–thermal ionization mass spectrometry dates retrieved by Rubidge et al., Botha et al., and Gastaldo et al. (37, 44, 80). The traditional (dashed red line) and associated PTB hypotheses for the Karoo Basin (37, 44) are also shown. Although traditionally associated with the PTB, the Daptocephalus–Lystrosaurus declivis Assemblage Zone boundary is defined by first appearances of co-occurring tetrapod assemblages, so its position relative to the three PTB hypotheses is unchanged. The Ripplemead member (*) has yet to be formalized by the South African Committee for Stratigraphy.Decades of research have demonstrated the richness of South Africa’s Karoo Basin fossil record, resulting in hundreds of stratigraphically well-documented tetrapod fossils across the PTT (37, 39, 56). This wealth of data has been used qualitatively to identify three extinction phases and an apparent early postextinction recovery phase (39, 45, 51). Furthermore, studies of Karoo community structure and function have elucidated the potential role of the extinction and subsequent recovery in breaking the incumbency of previously dominant clades, including synapsids (11, 57). Nevertheless, understanding patterns of faunal turnover and recovery during the PTT has been limited by the scarcity of quantitative investigations. Previous quantitative studies used coarsely sampled data (i.e., assemblage zone scale, 2 to 3 Ma time intervals) to identify low species richness immediately after the main extinction, potentially associated with multiple “boom and bust” cycles of primary productivity based on δ¹³C variation during the first 5 My of the Triassic (41, 58). However, many details of faunal dynamics in this interval remain unknown. Here, we investigate the dynamics of this major tetrapod extinction at an unprecedented time resolution (on the order of hundreds of thousands of years), using sample-standardized methods to quantify multiple aspects of regional change across the Cistecephalus, Daptocephalus, and Lystrosaurus declivis assemblage zones.     

Cryo-EM structure of Mycobacterium smegmatis DyP-loaded encapsulin

Encapsulins containing dye-decolorizing peroxidase (DyP)-type peroxidases are ubiquitous among prokaryotes, protecting cells against oxidative stress. However, little is known about how they interact and function. Here, we have isolated a native cargo-packaging encapsulin from Mycobacterium smegmatis and determined its complete high-resolution structure by cryogenic electron microscopy (cryo-EM). This encapsulin comprises an icosahedral shell and a dodecameric DyP cargo. The dodecameric DyP consists of two hexamers with a twofold axis of symmetry and stretches across the interior of the encapsulin. Our results reveal that the encapsulin shell plays a role in stabilizing the dodecameric DyP. Furthermore, we have proposed a potential mechanism for removing the hydrogen peroxide based on the structural features. Our study also suggests that the DyP is the primary cargo protein of mycobacterial encapsulins and is a potential target for antituberculosis drug discovery.

Compartmentalization is used by cells to overcome many difficult metabolic and physiological challenges (1). Eukaryotes employ membrane-bound organelles such as the mitochondrion (2); however, most prokaryotes rely on alternative proteinaceous compartments to achieve spatial control (3), one of which is the encapsulin nanocompartment.Encapsulins are newly identified nanocompartments but have already been applied in various scientific fields due to the unique structures (4, 5). It has been reported that more than 900 putative encapsulin systems in bacteria and archaea exist and are distributed across 15 bacterial and two archaeal phyla (6, 7), suggesting they are functionally diverse. Encapsulins are made of one type of shell protein, as opposed to several as is observed in many bacterial microcompartments (8, 9). The key feature of encapsulin systems is that cargo proteins can be specifically encapsulated and targeted to the encapsulin capsid interior, using a selective C-terminal sequence referred to as targeting peptides (TPs) (10). The functions of the nanocompartment are associated with the functions of its protein cargo. Many functionally diverse cargo proteins are associated with encapsulins, including dye-decolorizing peroxidases (DyPs) (11), ferritin-like proteins (FLP) (12), hydroxylamine oxidoreductase (HAO) (13), and cysteine desulfurases (14). Moreover, it has been shown that some encapsulin systems may possess multiple cargo proteins, which are made up of one core cargo protein and up to three secondary cargo proteins according to the TPs (6). Notably, a large proportion of native cargo proteins are DyP-type peroxidases, conferring the resistance of the cell to oxidative stress (6, 7, 11, 15–18). However, to date, the structural information on the cargo-encapsulated encapsulins is not yet available (SI Appendix, Table S1), and thus, little is known about the structural arrangement and mechanistic features of the cargo proteins loaded in the encapsulins.Actinobacteria harbors the largest number of encapsulin or encapsulin-like systems (6). DyP-containing encapsulins have already been reported from mycobacteria, including Mycobacterium smegmatis (15) and Mycobacterium tuberculosis (19). These have been considered as potential biomarkers to detect active tuberculosis (TB) (20). In the present study, we have isolated and characterized a DyP-loaded encapsulin system from M. smegmatis, which is commonly used as a model organism in studying the biology of the M. tuberculosis (21). We have determined its complete high-resolution structure by cryogenic electron microscopy (cryo-EM). Our results have revealed the interactions between the CFP-29 (a 29 kDa culture filtrate protein) shell and DyP cargo and a potential antioxidation mechanism. Our study also lays the foundation for the discovery of new diagnosis protocols and treatments of TB.