Cell

The common marmoset (Callithrix jacchus), a small monkey native to Brazil, has been used as a biomedical model in the United States (US) since the 1950s, yet the origins, genomic diversity, and population structure of current colonies remain poorly defined. Through the NIH Marmoset Coordinating Center, we registered and sampled most US research marmosets ([~]2,300 living animals) and assembled pedigrees and historical records for >10,000 individuals. We present a resource of >800 whole-genome sequences, largely from US colonies. These data reveal an unexpected population structure that predates the establishment of research colonies. Indeed, this population structure mirrors variation found in marmosets across Brazil. Leveraging sequenced families, we generate the first pedigree-based recombination map and improved estimates of de novo mutation processes for this species. Our insights into genetic diversity, structure, and inbreeding will guide colony management, inform disease modelling and strengthen the marmosets standing as a biomedical model. Further, this work demonstrates how coordinated efforts across colonies can enable a self-sustaining "living laboratory", supporting data sharing and well-powered studies beyond the reach of single institutions.

The population history of the Caribbeans first inhabitants has been challenging to reconstruct because few human remains are known from the regions earliest occupation which began around 6,000 years ago in Hispaniola, Cuba, and Puerto Rico. We generated genome-wide data from 19 individuals from Hispaniolas Samana Peninsula and focused on four who lived during the earliest pre-Ceramic "Lithic Age". Extending the Caribbean genetic record by more than a millennium to [~]4,400 calBP, we show that pre-Ceramic Age populations across Hispaniola and Cuba derive from a single ancestry source and document long-term genetic continuity across islands, with some local genetic structure within Hispaniola. Pre-Ceramic Age Caribbean ancestry shares most drift with populations from Central America and northern South America, although no sampled mainland group provides an adequate proxy. We infer very small effective community sizes, consistent with locally structured mating pools and little evidence of close-kin mating. These findings extend our understanding of Caribbean population history into its earliest phase. TeaserEarly Caribbean people shared ancestry across islands and lived in small, locally structured communities.

Transposition of all classes of transposable elements generates DNA intermediates that must be processed by the host to be effective. However, the mechanisms whereby transposons communicate with cellular DNA-processing machineries remain poorly investigated. Here, we provide convergent genetic and biochemical evidence that replicative transposition of the Tn3-family transposon Tn4430 is strictly coupled to replication of the target. Blocking target replication abolishes transposition, while blocking replication of the transposon donor molecule has no effect. Furthermore, the insertion preference of Tn4430 was found to be altered by the direction of replication and potential replication impediments within the target, suggesting a functional link between the integration mechanism and replication fork progression. In vitro, the transposase TnpA was found to specifically bind to fork-like DNA structures that mimic replication intermediates. Compared to linear DNA fragments, these structures are efficient substrates for TnpA-catalysed end joining. Strand transfer occurred immediately downstream of the fork, poising the transposon for replication. Together, the data suggest a mechanism in which the transposon targets DNA replication intermediates to directly recruit the host replication machinery at the time of transposition. This "replication hijacking" mechanism contrasts with classical "replication hiring" mechanisms during which replication is recruited after strand transfer. SIGNIFICANCE STATEMENTBacterial transposons of the Tn3 family constitute a threat for human health, being continuously involved in the emergence and spread of new antimicrobial resistances amongst pathogens. The success of these elements is based on their replicative mode of transposition allowing them to produce a new copy of themselves whenever they move. Here, we provide evidence that, rather than recruiting the host replication machinery after integration as is proposed in textbook models for replicative transposition, Tn3-family transposons directly transpose into ongoing replication intermediates. We propose that this "replication hijacking" mechanism provides a means of synchronizing transposition with cell replication activity, thus optimizing the movement of transposons with the expansion of bacterial populations. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=57 SRC="FIGDIR/small/728999v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@302040org.highwire.dtl.DTLVardef@1957f30org.highwire.dtl.DTLVardef@1d140b7org.highwire.dtl.DTLVardef@efeff0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Urinary tract infections (UTIs) are among the most common bacterial infections globally and create a large burden on the healthcare system. Uropathogenic Escherichia coli (UPEC) account for the majority of UTIs and increase the risk of recurrence. The standard treatment is antibiotics and, with the rise of multi-drug resistant UPEC lineages, there is a need for alternative treatments and prevention. Colicins, bacteriocins targeting and produced by E. coli, have previously been shown to inhibit the growth of pathogenic E. coli and are a promising alternative. Here, we engineer commensal Bacteroidaceae to secrete colicins via outer membrane vesicle (OMV) targeting signal peptides to suppress E. coli in the mouse gut. Secreted colicins were assessed for their ability to kill primary clinical isolate UPEC strains, including epidemic multi-drug resistant ST131 strains, along with other pathogenic and type strains. Specifically, secreted colicin E7, from Phocaeicola vulgatus fully eliminated of several UPEC strains in culture. In mice, P. vulgatus secreting colicin E7 prevented the extended colonization of two clinical UPEC strains and restored microbiome diversity. Together, this work shows the viability of secreted, heterologous antimicrobials from P. vulgatus as prophylactic treatment against the colonization of pathogenic E. coli utilizing cross-phylum antagonism in the gut. Significance StatementRecurrent urinary tract infections can be driven by intestinal reservoirs of uropathogenic Escherichia coli that are difficult to eliminate and increasingly recalcitrant to conventional antibiotic therapy. Here, we show that engineered gut symbionts from the Bacteroidaceae family can secrete targeted protein antibiotics to selectively kill these uropathogenic E. coli. Leveraging outer membrane vesicle-based secretion, we demonstrate that bacteriocin secretion can prevent gut colonization by clinically relevant pathogens, while preserving overall microbiome diversity. This work establishes a strategy for programmable, cross-phylum antimicrobial delivery within the gut microbiome, providing a potential alternative to conventional antibiotics for preventing recurrent infections and other enteric diseases.

Most of our understanding of endosymbiosis originates from the bacterial endosymbionts of multicellular, terrestrial hosts, which represent habitats with dramatically different selective pressures than inside a protist cell. Methanogenic archaea from the genus Methanocorpusculum are among the few known intracellular archaea and form unique symbioses with both animal and protist hosts, providing a unique opportunity to contrast symbiont evolution and function in very distinct host types. Here, we conducted phylo- and pangenomic analyses on 106 Methanocorpusculum strains originating from animal and ciliate hosts as well as environmental habitats. We recovered two divergent clades corresponding to animal gut-associated and intracellular ciliate-associated/environmental lineages and found that ciliate-associated and environmental Methanocorpusculum are virtually indistinguishable functionally and phylogenetically. Ciliate-associated symbionts retained broad biosynthetic capacity and encoded functions related to osmotic stress tolerance and adhesion within the host cell, while animal gut-associated symbionts exhibited patterns of genome streamlining and nutrient scavenging consistent with host supply and immune adaptation. Our findings illuminate how the contrasting selective pressures of protists and animal hosts have driven divergent evolutionary and functional strategies in congeneric archaeal symbionts.

Cancer immunotherapies rely on tumor-specific T cells, which arise endogenously in most patients with cancer, but can be low frequency and poorly functional. Methods to specifically identify, expand, and manipulate tumor-specific T cells at the rare frequencies found in peripheral blood would enable new immunotherapeutic strategies. Here, we demonstrate an approach to virally transduce polyclonal tumor-reactive T cells across any MHC haplotype and in the absence of knowing the cognate antigen. By generating lentiviral vectors that selectively transduce cells expressing 4-1BB (CD137), a marker of T cell activation, we can transduce antigen-specific T cells with user-defined genetic cargoes that can selectively expand and track individual clonotypes via single-cell sequencing. Anti-4-1BB lentiviruses (4-1BB LVs) encoding therapeutic cargoes can also enhance antigen-specific T cells to extend survival in a xenograft model of human melanoma and transduce tumor-infiltrating T cells from patients with ovarian cancer. Overall, the 4-1BB LV platform targets antigen-specific T cells in a manner agnostic to both the antigen and presenting MHC, with potential applications in adoptive cell therapy manufacturing and TCR identification. One Sentence SummaryEngineered lentiviral vectors targeting 4-1BB selectively activate, expand, and transduce antigen-specific T cells with immunomodulatory cargo.

Opsins underlie diverse physiological responses to light in animals. In the dark, most opsins bind the chromophore 11-cis retinal, which isomerizes to all-trans form upon light absorption, representing the initial key step in signaling. Maintenance of opsin function therefore requires continuous regeneration of the inactive, 11-cis-retinal-bound state. Here, we report a novel type of opsin, AtAntho2c, from a reef-building coral, whose active form, bound to all-trans retinal, can thermally revert to the initial dark state bound to 11-cis retinal. A cysteine residue in extracellular loop 2 region plays a key role in the self-regeneration ability. Using time-resolved and low-temperature spectroscopies, we identify two spectrally distinct photointermediates prior to the all-trans to 11-cis isomerization in AtAntho2c, whose formation rates and yields are found to vary depending on temperature and pH conditions. The active form of AtAntho2c activates Gi/o G protein, resulting in a transient and repeatable decrease in cellular cAMP levels upon repeated light stimulations, even in the absence of exogenous retinal in cultured cells. Furthermore, we confirm that cells expressing AtAntho2c exhibit membrane hyperpolarization via GIRK channel activation light-dependently. These properties highlight the potential of AtAntho2c as a versatile optogenetic actuator capable of repeatedly modulate Gi/o signaling without retinal supplementation. Significance StatementLight-sensitive proteins, opsins, form active states upon light absorption, leading to intracellular G protein signaling and various cellular outputs. The active states require specific enzymatic machinery or another photon absorption to regenerate inactive opsins ready to respond to repeated light stimuli and maintain continuous responsiveness. In our study, we identify and analyze a coral opsin of which the active state rapidly and autonomously reverts to the inactive state in the dark through thermal isomerization of the retinal chromophore within the opsin. This regeneration mechanism allows the opsin to respond to repeated light stimuli at high temporal resolution and maintain large signal amplitude without the need for exogenous retinal making this opsin potentially useful for developing versatile optogenetic tools.

Genetic variation is the raw material for evolution. One source of variation is chromosomal rearrangements, which can bring genes together and form genetic linkage. Rearrangements can also suppress recombination and gene flow, as in the case of sex chromosome evolution. We conducted the first population genomic study of the red harvester ant Pogonomyrmex barbatus to investigate genomic rearrangements that differentiate the lineages J1 and J2 in the "dependent-lineage system" (also known as "social hybridogenesis"). In this unusual reproductive system, males and females from different lineages mate to create hybrids, yet these hybrids develop into sterile offspring (workers), and so the two lineages remain reproductively isolated. We sequenced high-quality reference genomes for the two lineages to search for a potential explanation of the suppression of gene flow between them. Comparison of the two genome assemblies revealed multiple large-scale genomic rearrangements, all of which occurred in the J1 lineage. The rearrangements formed some of the largest J1 chromosomes, including the largest scaffold in the assembly that was formed by at least two translocation events and additional intra-chromosomal rearrangements. The translocations brought together 118 odorant receptor (OR) genes on this rearranged chromosome, 44 of which are 9-exon ORs, which are implicated in chemical communication in ants. We also identified an enrichment of transposable elements in a large synteny gap between the translocated segments. The discovery of multiple translocations that formed large rearranged chromosomes provides a potential explanation for the reproductive isolation between the pair of dependent lineages in this system, and opens the way for the study of the molecular genetic basis of an intriguing evolutionary phenomenon in these and in other ant lineages.

Plant viruses have evolved diverse strategies to facilitate their movement and survival within the host. Among them, geminiviruses co-opt host cellular machinery to replicate and disseminate. Traditionally, viral propagation has been associated with intercellular symplastic trafficking mediated by plasmodesmata and viral movement proteins. However, recent evidence demonstrated that the plant RNA virus turnip mosaic virus (TuMV) components are associated with extracellular vesicles (EVs). EVs are membrane-bound structures secreted to the extracellular space to potentially mediate several plant-pathogen interactions such as cross-kingdom RNA interference or the delivery of stress response proteins. In animals, EVs facilitate viral transmission both within the host and across species, but knowledge about their potential roles in plant viral infection is scarce. In this study, we demonstrate that EVs isolated from geminivirus-infected plants contain complete viral genomes and both capsid and viral movement proteins. Furthermore, these EV fractions were demonstrated to be infectious when mechanically inoculated onto naive plants. This discovery suggests that EVs may serve as alternative carriers for geminivirus components, enabling long-range transport or potentially modulating host immune responses, and highlights geminiviral capacity to transverse membrane boundaries, essential for circulative arbovirus propagation in their insect vectors. Significance StatementViruses are obligate intracellular parasites that shape ecological communities and crucially challenge animal and plant health worldwide. Conversely to animal viruses, plant-infecting viruses rely on plasmodesmata to disseminate through their host and establish systemic infection. Nonetheless, most plant viruses are insect-transmitted whose ecological cycle relies on their insect vector spread. Thus, strategies to cross continuous membrane barriers are essential for their dissemination and may be potentially conserved in plant hosts. Our discovery that infectious viral entities are associated with EVs reveals an alternative pathway for geminiviral movement within plant hosts that could facilitate vector transmission, challenging our long-standing understanding of plant virus biology and expanding the current conception of plant viral pathology.

Domain insertion is an established method to engineer ligand-mediated control of activity in protein scaffolds. Whether this strategy can be systematically applied to large, structured RNAs remains unclear. In this study, we investigated the feasibility of engineering ligand-activated splicing ribozymes (LASRs) from group I catalytic introns. Using domain-insertion profiling coupled with high-throughput screening, we mapped the nucleotide-resolution landscape of aptamer insertion across the ribozyme and identified sites that support robust ligand-dependent control. We showed LASRs function across multiple kingdoms of life, including diverse species of bacteria and even fungi, and can be used to regulate various genetic outputs. Finally, we integrated LASRs with a genetic recorder that writes information into ribosomal RNA, enabling sequencing-based recovery of intracellular chemical signals from microbial consortia. This work establishes LASRs as an RNA-based inducible control platform for sensing diverse chemical inputs, regulating the expression of diverse genes of interest, and recording intracellular information.

Programmable control of microbial gene expression by plant hosts could enable a new generation of precision agricultural biotechnology. Here, using O-methyl-L-tyrosine (OMY) as a model compound, we establish non-standard amino acids (nsAA) as a platform for plant-based control of associated microbial activity. We use genetic code expansion to engineer OMY-dependent control of protein synthesis in the soil bacterium Bacillus subtilis. Then, we engineer agronomically diverse plants, including Arabidopsis, tomato and poplar, to biosynthesize OMY. We show that plant-derived OMY can stimulate gene expression in both model and wild soil bacteria and demonstrate how inducible and tissue-specific expression of a single biosynthetic enzyme by the plant enables tight, on-demand control over microbial activity. This work establishes nsAAs as a tool for programming plant-microbe partnerships.

Understanding how naturally occurring genetic variation shapes human health and disease is critical for improving diagnosis and treatment strategies. The Mexican cavefish, Astyanax mexicanus, represents a powerful system for evolutionary medicine, enabling investigation of naturally evolved mechanisms of resilience to disease-related traits including diabetes, obesity, insomnia, and eye loss. Larval A. mexicanus, like zebrafish, are transparent, allowing whole-brain imaging, circuit mapping, and the generation of computationally derived atlases that precisely quantify neuroanatomical differences between surface and cave populations. Developing a molecular map of brain cell types provides a foundation for identifying evolved differences in neural circuits and physiology. Here, we present a single-cell atlas of the larval cavefish brain that reveals widespread divergence in the abundance and molecular signatures of neurons and glia. Our cell type map validates known neuroanatomical differences, including a reduction of the optic tectum and expansion of the pineal gland in cavefish. We uncover substantial changes in multiple glial cell classes that are linked to neural regulation of behavior, including microglia. Analysis of differential gene expression between surface and cavefish microglia revealed enhanced genes associated with synaptic pruning and clearance of neural debris, suggesting cavefish increased microglia activity to shape brain development. We also analyzed cell types that did not classify as canonical neurons or glia and identified notable divergence in transcriptomes and cell composition, including reduced meningeal fibroblasts in cavefish and substantial transcriptional changes related to phototransduction in non-visual photoreceptors within the pineal gland. Together, these findings provide a comprehensive atlas of cell type-specific gene expression differences between A. mexicanus surface and cavefish, establishing a platform for dissecting the molecular and cellular basis of evolved disease resilience in cavefish

Parasitism is one of the most widespread trophic strategies in nature, though its diversity and ecological distribution in marine ecosystems remain poorly characterized. Apicomplexa are a major clade of obligate parasites best known for medically important taxa, yet their diversity and distribution in the ocean is still largely unresolved. Here, we used metabarcoding data from the Tara expeditions to investigate the diversity, distribution, and environmental drivers of Apicomplexa across coral reef ecosystems and adjacent oceanic habitats. By integrating samples spanning planktonic communities, coral tissues, and marine sediments across multiple oceanic regions, we substantially expand the known phylogenetic breadth of marine apicomplexans. Although apicomplexans were generally low in relative abundance, they were widely distributed across marine environments. Community composition differed markedly among habitats. Corallicolid lineages were consistently associated with coral hosts, whereas planktonic samples harbored a greater diversity of apicomplexans, dominated by crustacean-associated gregarines. Sediments contained particularly high apicomplexan richness, including several poorly characterized groups. Capitalizing on the pan-Pacific transect of the expedition, we resolved biogeographic patterns in apicomplexan diversity across ocean basins: tropical regions showed the highest overall diversity, while polar environments contained distinct apicomplexan assemblages not detected in other ocean biomes. Together, these results highlight the extensive and previously underappreciated diversity of marine Apicomplexa and demonstrate that integrating multiple marine biomes is essential for resolving the phylogenetic and ecological breadth of parasitism in the ocean.

NLR immune receptor networks consist of expanded disease resistance proteins (sensor NLRs) that signal via core executors of immunity known as helper NLRs. Although some sensor NLRs are thought to activate their cognate helpers via an activation-and-release mechanism, the structural basis of sensor-helper communication remains poorly understood. Here, we identify and validate sensor-helper NLR interfaces that are critical for immune activation in the NRC network of coiled-coil NLR immune receptors. Using AlphaFold 3 we predicted a high confidence model between the virus resistance protein Rx and its helper NLR NRC2. We validated the interfaces by loss and gain-of-function mutagenesis, including reconstituting a critical salt bridge through reciprocal mutations. We showed that these interfaces are conserved across the NRC network of asterid plants despite over 120 million years of divergence and validated the sensor-NRC interfaces within the common lettuce network. Structure-guided bioengineering of a lettuce sensor NLR enabled expansion of its NRC helper compatibility profile. These results are consistent with the activation-and-release model and point to bioengineering sensor-helper specificity in economically important crop species.

Coronavirus genome encapsidation depends on cis-acting RNA elements that interact with viral structural proteins. While such packaging signals have been characterized in several coronaviruses, their definition in SARS-CoV-2 remains incomplete. Using synthetic defective SARS-CoV-2 genomes, we identify a 667-nucleotide region within the nsp15 coding sequence that preferentially binds SARS-CoV-2 nucleoprotein and enhances the accumulation of defective viral genomes both in vitro and in vivo. Sequential and targeted deletion analyses further delineate candidate RNA secondary structures within this region that contribute to this enrichment. These structures show similarity to elements within the putative packaging signal of SARS-CoV but are not conserved across other coronaviruses. Together, these findings support the presence of a structured RNA element within nsp15 that contributes to SARS-CoV-2 genome encapsidation and provide a framework for further structural and functional dissection of coronavirus packaging signals. IMPORTANCEThis study identifies a 667-nt region within the SARS-CoV-2 nsp15 coding sequence that binds nucleoprotein and promotes accumulation of defective viral genomes, revealing a previously unrecognized contributor to genome encapsidation. Mapping of candidate RNA structures within this region links SARS-CoV-2 packaging activity to conserved structural features observed in SARS-CoV, while highlighting key differences from other coronaviruses. These findings refine understanding of cis-acting packaging signals in SARS-CoV-2 and provide a foundation for further structural and functional analysis of coronavirus genome encapsidation. O_FIG O_LINKSMALLFIG WIDTH=177 HEIGHT=200 SRC="FIGDIR/small/721935v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1b64611org.highwire.dtl.DTLVardef@1b21b7borg.highwire.dtl.DTLVardef@2a68b5org.highwire.dtl.DTLVardef@405fe1_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACTC_FLOATNO A part of the nsp15 coding sequence of SARS-CoV-2 promotes efficient transmission of defective viral genomes in vitro and in vivo. Using a sequential deletion library and targeted deletions within this region we identify RNA structures that may function as packaging signals. C_FIG

Venoms are powerful weapons that shape ecological interactions across the animal kingdom. They also have high medical importance, causing thousands of human fatalities annually, while offering a rich resource for drug discovery. Despite this, considerable logistical, safety and ethical challenges mean only a fraction of the worlds venoms have been profiled quantitatively. Natural history collections offer opportunities to greatly expand our knowledge of venom systems. We used quantitative proteomic mass spectrometry on preserved venom glands, spanning 0-57 years in age, from 37 venomous snake species (32 elapids and 5 viperids), alongside fresh venom samples from most of the same taxa. Preserved glands and fresh venoms from the same species showed strong concordance in venom composition across multiple metrics. Critically, specimen age did not degrade data quality, indicating that decades-old material yields reliable quantitative profiles, opening the possibility for vast quantities of existing museum specimens to be used. When we integrate our data with published profiles, we confirm known elapid vs viper broad diversity patterns while revealing substantial Australasian elapid venom diversity. Our findings demonstrate the potential of natural history collections as a vast, largely untapped biochemical archive for high-throughput "museum venomics," enabling evolutionary and temporal analyses of venom diversity at unprecedented scale.

Microbes that remain uncultivated occupy nearly every ecosystem on the planet; this is particularly true in soils, where despite their prevalence, the roles of rarely cultivated microbes in driving biogeochemical cycles and ecosystem function remain poorly explored. We combine metagenome-informed substrate selection with enrichment sub-communities to generate reduced-complexity communities that preserve co-occurrence and expand experimental access to underrepresented soil lineages without requiring prior isolation of each member. Carbohydrate-active enzyme (CAZyme) profiles from soil-derived genomes were used to select carbon compounds predicted to enrich difficult to culture taxa, including members of the phylum Acidobacteriota. Based on 16S rRNA amplicon sequencing, we reproducibly enriched Terriglobus (Acidobacteriota) on multiple metagenome-guided substrates. Select communities with consistent presence and varying abundance of Terriglobus were passaged in a longitudinal design to generate 89 metagenomes; genus-level profiling revealed that community composition varied between biological replicates but remained consistent within replicates over time, providing diverse Acidobacteriota-containing configurations for downstream analysis. Association network inference identified a core set of co-occurring taxa that positively tracked with Terriglobus across the longitudinal series. In parallel, the substrate-guided approach led to isolation of a novel Terriglobus species, the first cultured representative of its GTDB species cluster. Together, these results establish a generalizable strategy for generating communities enriched with rarely cultivated taxa, yielding tractable systems for studying microbial interactions and community assembly in soil.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large category of natural products and a promising source for new medicines and applications in biotechnology. Their extraordinary diversity stems from the sequences of ribosomally synthesized precursor peptides and the wide repertoire of biosynthetic proteins that post-translationally modify and process the peptides. However, known precursor peptides and biosynthetic events have been characterized as soluble and occurring within aqueous environments in which RiPPs encounter the membrane primarily for secretion via transporters. Here, we report that the cell membrane is the central setting for the biosynthesis of TMcins, a recently discovered RiPP class with antimicrobial activity that contains a transmembrane helix (TMH) in the precursor peptide and mature product. We show that the ribosomally synthesized precursor TmcA is integrated in the producing cell membrane. We then uncovered the biosynthetic roles of gene products encoded on the TMcin biosynthetic gene cluster by integrating structure-prediction with an inducible TMcin biosynthesis platform. All TMcin post-translational modifications occurred in the membrane, for which three of four events were performed by intramembranous biosynthetic proteins. Finally, we assign an escort function to a previously uncharacterized membrane protein and provide insights into its evolution. Thus, our characterization of TMcin expands the setting for RiPP biosynthesis and provides a model for the biosynthesis of membrane-localized peptide natural products. Significance StatementCells modify peptides to make natural products called RiPPs that exhibit a wide range of activities. Many RiPPs are antimicrobial and are important sources for discovering new drugs. Known RiPPs are produced by biosynthetic enzymes in aqueous environments. We characterized the recently discovered TMcin class of RiPPs that possess a transmembrane helix and are found across Gram-positive bacteria. Using a Staphylococcus aureus strain that naturally produces TMcin, we show that TMcin biosynthesis occurs in the membrane and elucidate the steps of TMcin production. This work establishes a model for understanding RiPP biosynthesis within a lipid environment that broadens our understanding of RiPPs, enables discoveries of new RiPPs, and provides a basis for the rational engineering of TMcins.

Understanding how crops respond to environmental variation is crucial for biodiversity conservation and food security. Cucurbita pepo (pumpkin, squash, gourd) is one of the first domesticated crop species and exhibits remarkable phenotypic and ecological diversity, making it a powerful system for investigating the genomic basis of adaptation. Here, we constructed a graph-based C. pepo pangenome using nine chromosome-level assemblies and identified 229,431 high-confidence structural variants (SVs) that were genotyped across 206 wild and cultivated accessions. Our results demonstrate that C. pepo underwent parallel domestication, with two deeply diverged gene pools independently giving rise to the pepo and ovifera cultivated lineages, followed by expansion into diverse environments that produced strong signatures of differentiation largely mediated by young adaptive alleles. Single-nucleotide polymorphisms (SNPs) and SVs contribute complementary dimensions of environmental responsiveness. Biogeographical modeling predicts continued range contraction of wild relatives and elevated genetic offset in Eastern North American populations under projected future climates. Populations with higher genetic load harbor fewer adaptive variants and exhibit greater predicted maladaptation, indicating that deleterious mutations constrain adaptive potential. These findings highlight how genomic variants, adaptive diversity, and genetic load together shape environmental adaptation and inform conservation and crop improvement.

Ca2+ signaling mediates rapid cellular responses across eukaryotes, but its spatiotemporal dynamics remain largely inaccessible in many genetically less tractable microbial lineages. Oomycete plant pathogens, including Phytophthora species, undergo rapid transitions between motile, encysted, germinating, and invasive stages, yet the organization of Ca2+ dynamics during these transitions is poorly understood. Here, we adapt the genetically encoded ratiometric biosensor MatryoshCaMP8s for in vivo calcium imaging in Phytophthora palmivora. The reporter is stably expressed without major detectable effects on sporulation or virulence and reports rapid ratiometric responses to cold shock and calcimycin/A23187. Using live-cell imaging, we uncover stage-specific Ca2+ dynamics across the pre-infective and early infection cycle. Sporangia approaching zoospore release display spatially heterogeneous Ca2+ transients, newly formed cysts occasionally exhibit Ca2+ transients, and germinating cysts show recurrent Ca2+ transients at the germ tube tip. Similar sharp ratiometric pulses occur during early plant infection, indicating that polarized Ca2+ transients are not restricted to in vitro germination but recur at the host surface. Together, our work establishes live ratiometric calcium imaging in oomycetes, reveals polarized Ca2+ transients as recurrent signatures of developmental transitions and early host colonization, and opens the way to mechanistic dissection of signaling, polarity, and infection in a major group of plant pathogens.