Tracking magma pathways and surface faulting in the Southwest Rift Zone and the Koa'e fault system (Kilauea volcano, Hawai 'i) using photogrammetry and structural observations.

Volcanic islands are often subject to flank instability, resulting from a combination of magmatic intrusions along rift zones and gravitational spreading causing extensional faulting at the surface. Here, we study the Koa'e fault system (KFS), located south of the summit caldera of Kilauea volcano in Hawai'i, one of the most active volcanoes on Earth, prone to active faulting, episodic dike intrusions, and flank instability. Two rift zones and the KFS are major structures controlling volcanic flank instability and magma propagation. Although several magmatic intrusions occurred over the KFS, the link between these faults, two nearby rift zones and the flank instability, is still poorly studied. To better characterize the KFS and its structural linkage with the surrounding fault and rift zones, we performed a detailed structural analysis of the extensional fault system, coupled with a helicopter photogrammetric survey, covering part of the south flank of Kilauea. We generated a high-resolution DEM (~ 8 cm) and orthomosaic (~ 4 cm) to map the fracture field in detail. We also collected ~ 1000 ground structural measurements of extensional fractures during our three field missions (2019, 2022, and 2023). We observed many small, interconnected grabens, monoclines, rollover structures, and en-echelon fractures that were in part previously undocumented. We estimate the cumulative displacement rate across the KFS during the last 600 ~ 700 years and found a decrease toward the west of the horizontal component from 2 to 6 cm per year, consistent with GNSS data. Integrating morphology observations, fault mapping, and kinematic measurements, we propose a new kinematic model of the upper part of the Kilauea's south flank, suggesting a clockwise rotation and a translation of a triangular wedge. This wedge is bordered by the extensional structures (ERZ, SWRZ, and the KFS), largely influenced by gravitational spreading. These findings illustrate a structural linkage between the two rift zones and the KFS, the latter being episodically affected by dike intrusions. Supplementary Information: The online version contains supplementary material available at 10.1007/s00445-024-01735-7.

Improving consistency in estimating future health burdens from environmental risk factors: Case study for ambient air pollution.

Future changes in exposure to risk factors should impact mortality rates and population. However, studies commonly use mortality rates and population projections developed exogenously to the health impact assessment model used to quantify future health burdens attributable to environmental risks that are therefore invariant to projected exposure levels. This impacts the robustness of many future health burden estimates for environmental risk factors. This work describes an alternative methodology that more consistently represents the interaction between risk factor exposure, population and mortality rates, using ambient particulate air pollution (PM2.5) as a case study. A demographic model is described that estimates future population based on projected births, mortality and migration. Mortality rates are disaggregated between the fraction due to PM2.5 exposure and other factors for a historic year, and projected independently. Accounting for feedbacks between future risk factor exposure and population and mortality rates can greatly affect estimated future attributable health burdens. The demographic model estimates much larger PM2.5-attributable health burdens with constant 2019 PM2.5 (∼10.8 million deaths in 2050) compared to a model using exogenous population and mortality rate projections (∼7.3 million), largely due to differences in mortality rate projection methods. Demographic model-projected PM2.5-attributable mortality can accumulate substantially over time. For example, ∼71 million more people are estimated to be alive in 2050 when WHO guidelines (5 µg m-3) are achieved compared to constant 2019 PM2.5 concentrations. Accounting for feedbacks is more important in applications with relatively high future PM2.5 concentrations, and relatively large changes in non-PM2.5 mortality rates.

The condition-dependence of male genital size and shape.

The male genitals of internal fertilisers evolve rapidly and divergently, and sexual selection is generally responsible for this. Many sexually selected traits are condition-dependent-with their expression dependent upon the resources available to be allocated to them-as revealed by genetic or environmental manipulations of condition. However, it is not clear whether male genitals are also condition-dependent. Here we manipulate condition in two ways (via inbreeding and diet) to test the condition-dependence of the genital arch of Drosophila simulans. We found that genital size but not genital shape suffered from inbreeding depression, whereas genital size and shape were affected by dietary manipulation of condition. The differential effects of these treatments likely reflect underlying genetic architecture that has been shaped by past selection: inbreeding depression is only expected when traits have a history of directional selection, while diet impacts traits regardless of historical selection. Nonetheless, our results suggest genitals can be condition-dependent like other sexually selected traits.

Convergence of coronary artery disease genes onto endothelial cell programs.

Linking variants from genome-wide association studies (GWAS) to underlying mechanisms of disease remains a challenge1-3. For some diseases, a successful strategy has been to look for cases in which multiple GWAS loci contain genes that act in the same biological pathway1-6. However, our knowledge of which genes act in which pathways is incomplete, particularly for cell-type-specific pathways or understudied genes. Here we introduce a method to connect GWAS variants to functions. This method links variants to genes using epigenomics data, links genes to pathways de novo using Perturb-seq and integrates these data to identify convergence of GWAS loci onto pathways. We apply this approach to study the role of endothelial cells in genetic risk for coronary artery disease (CAD), and discover 43 CAD GWAS signals that converge on the cerebral cavernous malformation (CCM) signalling pathway. Two regulators of this pathway, CCM2 and TLNRD1, are each linked to a CAD risk variant, regulate other CAD risk genes and affect atheroprotective processes in endothelial cells. These results suggest a model whereby CAD risk is driven in part by the convergence of causal genes onto a particular transcriptional pathway in endothelial cells. They highlight shared genes between common and rare vascular diseases (CAD and CCM), and identify TLNRD1 as a new, previously uncharacterized member of the CCM signalling pathway. This approach will be widely useful for linking variants to functions for other common polygenic diseases.

Impaired islet function with normal exocrine enzyme secretion is consistent across the head, body, and tail pancreas regions in type 1 diabetes.

Histopathological heterogeneity in human pancreas has been well documented; however, functional evidence at the tissue level is scarce. Herein we investigated in situ glucose-stimulated islet and carbachol-stimulated acinar cell secretion across the pancreas head (PH), body (PB), and tail (PT) regions in no diabetes (ND, n=15), single islet autoantibody-positive (1AAb+, n=7), and type 1 diabetes donors (T1D, <14 months duration, n=5). Insulin, glucagon, pancreatic amylase, lipase, and trypsinogen secretion along with 3D tissue morphometrical features were comparable across the regions in ND. In T1D, insulin secretion and beta-cell volume were significantly reduced within all regions, while glucagon and enzymes were unaltered. Beta-cell volume was lower despite normal insulin secretion in 1AAb+, resulting in increased volume-adjusted insulin secretion versus ND. Islet and acinar cell secretion in 1AAb+ were consistent across PH, PB and PT. This study supports low inter-regional variation in pancreas slice function and potentially, increased metabolic demand in 1AAb+.

Nucleoid-associated proteins shape the global protein occupancy and transcriptional landscape of a clinical isolate of Vibrio cholerae.

Vibrio cholerae, the causative agent of the diarrheal disease cholera, poses an ongoing health threat due to its wide repertoire of horizontally acquired elements (HAEs) and virulence factors. New clinical isolates of the bacterium with improved fitness abilities, often associated with HAEs, frequently emerge. The appropriate control and expression of such genetic elements is critical for the bacteria to thrive in the different environmental niches it occupies. H-NS, the histone-like nucleoid structuring protein, is the best studied xenogeneic silencer of HAEs in gamma-proteobacteria. Although H-NS and other highly abundant nucleoid-associated proteins (NAPs) have been shown to play important roles in regulating HAEs and virulence in model bacteria, we still lack a comprehensive understanding of how different NAPs modulate transcription in V. cholerae. By obtaining genome-wide measurements of protein occupancy and active transcription in a clinical isolate of V. cholerae, harboring recently discovered HAEs encoding for phage defense systems, we show that a lack of H-NS causes a robust increase in the expression of genes found in many HAEs. We further found that TsrA, a protein with partial homology to H-NS, regulates virulence genes primarily through modulation of H-NS activity. We also identified a few sites that are affected by TsrA independently of H-NS, suggesting TsrA may act with diverse regulatory mechanisms. Our results demonstrate how the combinatorial activity of NAPs is employed by a clinical isolate of an important pathogen to regulate recently discovered HAEs. Importance: New strains of the bacterial pathogen Vibrio cholerae, bearing novel horizontally acquired elements (HAEs), frequently emerge. HAEs provide beneficial traits to the bacterium, such as antibiotic resistance and defense against invading bacteriophages. Xenogeneic silencers are proteins that help bacteria harness new HAEs and silence those HAEs until they are needed. H-NS is the best-studied xenogeneic silencer; it is one of the nucleoid-associated proteins (NAPs) in gamma-proteobacteria and is responsible for the proper regulation of HAEs within the bacterial transcriptional network. We studied the effects of H-NS and other NAPs on the HAEs of a clinical isolate of V. cholerae. Importantly, we found that H-NS partners with a small and poorly characterized protein, TsrA, to help domesticate new HAEs involved in bacterial survival and in causing disease. Proper understanding of the regulatory state in emerging isolates of V. cholerae will provide improved therapies against new isolates of the pathogen.

A Vibrio cholerae viral satellite maximizes its spread and inhibits phage by remodeling hijacked phage coat proteins into small capsids.

Phage satellites commonly remodel capsids they hijack from the phages they parasitize, but only a few mechanisms regulating the change in capsid size have been reported. Here, we investigated how a satellite from Vibrio cholerae, phage-inducible chromosomal island-like element (PLE), remodels the capsid it has been predicted to steal from the phage ICP1 (Netter et al., 2021). We identified that a PLE-encoded protein, TcaP, is both necessary and sufficient to form small capsids during ICP1 infection. Interestingly, we found that PLE is dependent on small capsids for efficient transduction of its genome, making it the first satellite to have this requirement. ICP1 isolates that escaped TcaP-mediated remodeling acquired substitutions in the coat protein, suggesting an interaction between these two proteins. With a procapsid-like particle (PLP) assembly platform in Escherichia coli, we demonstrated that TcaP is a bona fide scaffold that regulates the assembly of small capsids. Further, we studied the structure of PLE PLPs using cryogenic electron microscopy and found that TcaP is an external scaffold that is functionally and somewhat structurally similar to the external scaffold, Sid, encoded by the unrelated satellite P4 (Kizziah et al., 2020). Finally, we showed that TcaP is largely conserved across PLEs. Together, these data support a model in which TcaP directs the assembly of small capsids comprised of ICP1 coat proteins, which inhibits the complete packaging of the ICP1 genome and permits more efficient packaging of replicated PLE genomes.

What factors influence the rediscovery of lost tetrapod species?

We created a database of lost and rediscovered tetrapod species, identified patterns in their distribution and factors influencing rediscovery. Tetrapod species are being lost at a faster rate than they are being rediscovered, due to slowing rates of rediscovery for amphibians, birds and mammals, and rapid rates of loss for reptiles. Finding lost species and preventing future losses should therefore be a conservation priority. By comparing the taxonomic and spatial distribution of lost and rediscovered tetrapod species, we have identified regions and taxa with many lost species in comparison to those that have been rediscovered-our results may help to prioritise search effort to find them. By identifying factors that influence rediscovery, we have improved our ability to broadly distinguish the types of species that are likely to be found from those that are not (because they are likely to be extinct). Some lost species, particularly those that are small and perceived to be uncharismatic, may have been neglected in terms of conservation effort, and other lost species may be hard to find due to their intrinsic characteristics and the characteristics of the environments they occupy (e.g. nocturnal species, fossorial species and species occupying habitats that are more difficult to survey such as wetlands). These lost species may genuinely await rediscovery. However, other lost species that possess characteristics associated with rediscovery (e.g. large species) and that are also associated with factors that negatively influence rediscovery (e.g. those occupying small islands) are more likely to be extinct. Our results may foster pragmatic search protocols that prioritise lost species likely to still exist.

The Social Cost of Ozone-Related Mortality Impacts From Methane Emissions.

Atmospheric methane directly affects surface temperatures and indirectly affects ozone, impacting human welfare, the economy, and environment. The social cost of methane (SC-CH4) metric estimates the costs associated with an additional marginal metric ton of emissions. Current SC-CH4 estimates do not consider the indirect impacts associated with ozone production from changes in methane. We use global model simulations and a new BenMAP webtool to estimate respiratory-related deaths associated with increases in ozone from a pulse of methane emissions in 2020. By using an approach consistent with the current SC-CH4 framework, we monetize and discount annual damages back to present day values. We estimate that the methane-ozone mechanism is attributable to 760 (95% CI: 330-1200) respiratory-related deaths per million metric tons of methane globally, for a global net present damage of $1800/mT (95% CI: $760-$2800/Mt CH4; 2% Ramsey discount rate); this would double the current SC-CH4 if included. These physical impacts are consistent with recent studies, but comparing direct costs is challenging. Economic damages are sensitive to uncertainties in the exposure and health risks associated with tropospheric ozone, assumptions about future projections of NOx emissions, socioeconomic conditions, and mortality rates, monetization parameters, and other factors. Our estimates are highly sensitive to uncertainties in ozone health risks. We also develop a reduced form model to test sensitivities to other parameters. The reduced form tool runs with a user-supplied emissions pulse, as well as socioeconomic and precursor projections, enabling future integration of the methane-ozone mechanism into the SC-CH4 modeling framework.

Adaptation to bile and anaerobicity limits Vibrio cholerae phage adsorption.

Bacteriophages (viruses of bacteria) play a pivotal role in shaping both the evolution and dynamics of bacterial populations. Bacteria employ arsenals of genetically encoded phage defense systems, but can alternatively achieve protection by changing the availability of cellular resources that phages rely on for propagation. These physiological changes are often adaptive responses to unique environmental signals. The facultative pathogen Vibrio cholerae adapts to both aquatic and intestinal environments with niche-specific physiological changes that ensure its evolutionary success in such disparate settings. In both niches, V. cholerae is susceptible to predation by lytic phages like ICP1. However, both phages and susceptible bacterial hosts coexist in nature, indicating that environmental cues may modulate V. cholerae cell state to protect against phage infection. This work explores one such modification in response to the intestine-specific signals of bile and anaerobicity. We found that V. cholerae grown in these conditions reduces O1-antigen decoration on its outer membrane lipopolysaccharide. Because the O1-antigen is an essential moiety for ICP1 phage infection, we investigated the effect of partial O1-antigen depletion as a mechanism of phage defense and observed that O1-depletion limits phage adsorption. We identified mechanistic contributions to O1-depletion, including the essentiality of a weak acid tolerance system for O1 production at low pH and alterations in transcriptional profiles indicating limitations in resources for O1-biosynthesis. This analysis illustrates a complex interplay between signals relevant to the intestinal environment and bacterial physiology that provides V. cholerae with protection from phage predation. IMPORTANCE Vibrio cholerae is the bacterial pathogen responsible for cholera, a diarrheal disease that impacts people in areas without access to potable water. In regions that lack such infrastructure, cholera represents a large proportion of disease outbreaks. Bacteriophages (phages, viruses that infect bacteria) have recently been examined as potential therapeutic and prophylactic agents to treat and prevent bacterial disease outbreaks like cholera due to their specificity and stability. This work examines the interaction between V. cholerae and vibriophages in consideration for a cholera prophylaxis regimen (M. Yen, L. S. Cairns, and A. Camilli, Nat Commun 8:14187, 2017, https://doi.org/10.1038/ncomms14187) in the context of stimuli found in the intestinal environment. We discover that common signals in the intestinal environment induce cell surface modifications in V. cholerae that also restrict some phages from binding and initiating infection. These findings could impact considerations for the design of phage-based treatments, as phage infection appears to be limited by bacterial adaptations to the intestinal environment.

Parkland Trauma Index of Mortality in Orthopaedic Trauma Patients: An Initial Report.

OBJECTIVES: The extent and timing of surgery in severely injured patients remains an unsolved problem in orthopaedic trauma. Different laboratory values or scores have been used to try to predict mortality and estimate physiological reserve. The Parkland Trauma Index of Mortality (PTIM) has been validated as an electronic medical record-integrated algorithm to help with operative timing in trauma patients. The aim of this study was to report our initial experience with PTIM and how it relates to other scores. METHODS: A retrospective chart review of level 1 and level 2 trauma patients admitted to our institution between December 2020 and November 2022 was conducted. Patients scored with PTIM with orthopaedic injuries were included in this study. Exclusion criteria were patients younger than 18 years. RESULTS: Seven hundred seventy-four patients (246 female patients) with a median age of 40.5 (18-101) were included. Mortality was 3.1%. Patients in the PTIM high-risk category (≥0.5) had a 20% mortality rate. The median PTIM was 0.075 (0-0.89) and the median Injury Severity Score (ISS) was 9.0 (1-59). PTIM (P < 0.001) and ISS (P < 0.001) were significantly lower in surviving patients. PTIM was mentioned in 7.6% of cases, and in 1.7% of cases, providers indicated an action in response to the PTIM. PTIM and ISS were significantly higher in patients with documented PTIM. CONCLUSION: PTIM is better at predicting mortality compared with ISS. Our low rate of PTIM documentation in provider notes highlights the challenges of implementing a new algorithm. LEVEL OF EVIDENCE: Level III, retrospective cohort study.

Novobiocin blocks nucleic acid binding to Polθ and inhibits stimulation of its ATPase activity.

Polymerase theta (Polθ) acts in DNA replication and repair, and its inhibition is synthetic lethal in BRCA1 and BRCA2-deficient tumor cells. Novobiocin (NVB) is a first-in-class inhibitor of the Polθ ATPase activity, and it is currently being tested in clinical trials as an anti-cancer drug. Here, we investigated the molecular mechanism of NVB-mediated Polθ inhibition. Using hydrogen deuterium exchange-mass spectrometry (HX-MS), biophysical, biochemical, computational and cellular assays, we found NVB is a non-competitive inhibitor of ATP hydrolysis. NVB sugar group deletion resulted in decreased potency and reduced HX-MS interactions, supporting a specific NVB binding orientation. Collective results revealed that NVB binds to an allosteric site to block DNA binding, both in vitro and in cells. Comparisons of The Cancer Genome Atlas (TCGA) tumors and matched controls implied that POLQ upregulation in tumors stems from its role in replication stress responses to increased cell proliferation: this can now be tested in fifteen tumor types by NVB blocking ssDNA-stimulation of ATPase activity, required for Polθ function at replication forks and DNA damage sites. Structural and functional insights provided in this study suggest a path for developing NVB derivatives with improved potency for Polθ inhibition by targeting ssDNA binding with entropically constrained small molecules.

Using Affinity Pulldown Assays to Study Protein-Protein Interactions of Human NEIL1 Glycosylase and the Checkpoint Protein RAD9-RAD1-HUS1 (9-1-1) Complex.

Affinity pulldown is a powerful technique to discover novel interaction partners and verify a predicted physical association between two or more proteins. Pulldown assays capture a target protein fused with an affinity tag and analyze the complexed proteins. Here, we detail methods of pulldown assays for two high-affinity peptide fusion tags, Flag tag (DYKDDDDK) and hexahistidine tag (6xHis), to study protein-protein interactions of human NEIL1 glycosylase and the checkpoint protein complex RAD9-RAD1-HUS1 (9-1-1). We uncover unique interactions between 9-1-1 and NEIL1, which suggest a possible inhibitory role of the disordered, phosphorylated C-terminal region of RAD9 in regulating NEIL1 activity in base excision repair through lack of physical association of 9-1-1 and NEIL1.

Nuclease genes occupy boundaries of genetic exchange between bacteriophages.

Homing endonuclease genes (HEGs) are ubiquitous selfish elements that generate targeted double-stranded DNA breaks, facilitating the recombination of the HEG DNA sequence into the break site and contributing to the evolutionary dynamics of HEG-encoding genomes. Bacteriophages (phages) are well-documented to carry HEGs, with the paramount characterization of HEGs being focused on those encoded by coliphage T4. Recently, it has been observed that the highly sampled vibriophage, ICP1, is similarly enriched with HEGs distinct from T4's. Here, we examined the HEGs encoded by ICP1 and diverse phages, proposing HEG-driven mechanisms that contribute to phage evolution. Relative to ICP1 and T4, we found a variable distribution of HEGs across phages, with HEGs frequently encoded proximal to or within essential genes. We identified large regions (> 10kb) of high nucleotide identity flanked by HEGs, deemed HEG islands, which we hypothesize to be mobilized by the activity of flanking HEGs. Finally, we found examples of domain swapping between phage-encoded HEGs and genes encoded by other phages and phage satellites. We anticipate that HEGs have a larger impact on the evolutionary trajectory of phages than previously appreciated and that future work investigating the role of HEGs in phage evolution will continue to highlight these observations.

Deciphering the potential of plasma cell-free metagenomic next-generation sequencing using the Karius test.

PURPOSE OF REVIEW: Plasma cell-free metagenomic next-generation sequencing (cf-mNGS) is increasingly employed for the diagnosis of infection, but a consensus for optimal use has not been established. This minireview focuses on the commercially available Karius Test and is aimed at local leaders seeking to understand the complexities of cf-mNGS to make informed test utilization policies and better interpret results. RECENT FINDINGS: Recent retrospective studies have reported how the Karius Test was applied at their institutions and identified areas of potential patient benefit. In addition, substantive studies have reported how this test performs in specific indications, particularly invasive fungal disease, endovascular infection and lower respiratory infection. SUMMARY: Successfully integrating plasma cf-mNGS requires careful assessment of performance in the specific applications and patient populations in which it is used. Individual institutions must independently evaluate implementation strategies and determine where diagnostic yields outweigh the potential pitfalls.

Genomic profile of Pancoast syndrome due to hepatocellular carcinoma: A case report.

Hepatocellular carcinoma (HCC) is a common cancer and is frequently diagnosed at a late and unresectable stage with limited effective treatment options. Here, we present the fifth reported case of a 77 year-old male with metastatic HCC presenting as a symptomatic superior sulcus lung tumor and discuss the genomic profile of this rare presentation of HCC for the first time, which included multiple classic mutations in HCC such as TERT, TP53, and WNT/ß-catenin signaling as well as in the DNA repair gene ATM. The patient was treated with palliative radiotherapy to the Pancoast tumor followed by atezolizumab plus bevacizumab and passed away 6 months after diagnosis. This rare case highlights the need for effective treatment in aggressive and unresectable HCC and the utility of early genomic studies to allow for targeted therapy such as poly (ADP-ribose) polymerase (PARP)-inhibitors.

Risk Factors and Characteristics of Recalcitrant Osteomyelitis After Initial Surgical and Antibiotic Treatment.

OBJECTIVES: To evaluate the injury, patient, and microbiological characteristics that place patients at risk for recalcitrant fracture-related infection and osteomyelitis despite appropriate initial treatment. DESIGN: Retrospective chart review. SETTING: Three level I trauma centers. PATIENTS AND PARTICIPANTS: Two hundred and fifty-seven patients undergoing surgical debridement and antibiotic therapy for osteomyelitis from 2003 to 2019. MAIN OUTCOME MEASUREMENTS: Patients were categorized as having undergone serial bone debridement if they had 2 separate procedures a minimum of 6 weeks apart with a full course of appropriate antibiotics in between. Patient records were reviewed for age, injury location, body mass index, smoking status, comorbidities, and culture results including the presence of multidrug-resistant organisms and culture-negative osteomyelitis. RESULTS: A total of 257 patients were identified; 49% (n = 125) had a successful single course of treatment, and 51% (n = 132) required repeat debridement for recalcitrant osteomyelitis. At the index treatment for osteomyelitis, the most common organisms in both groups were methicillin-resistant (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA). There was no significant difference in incidence of polymicrobial infection between the 2 groups (25% vs. 20%, P = 0.49). The most common organisms cultured at the time of repeat saucerization remained MRSA and MSSA; however, the same organism was cultured from both the index and repeat procedures in only 28% (n = 37) of cases. Diabetic patients, intravenous drug use status, delay to diagnosis, and open fractures of the lower leg are independent risk factors for failure of initial treatment of posttraumatic osteomyelitis. CONCLUSIONS: Successful eradication of fracture-related infection and posttraumatic osteomyelitis is difficult and fails 51% of the time despite standard surgical and antimicrobial therapy. Although MRSA and MSSA remain the most common organisms cultured, patients who fail initial treatment for osteomyelitis often do not culture the same organisms as those obtained at the index procedure. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

A Vibrio cholerae viral satellite maximizes its spread and inhibits phage by remodeling hijacked phage coat proteins into small capsids.

Phage satellites commonly remodel capsids they hijack from the phages they parasitize, but only a few mechanisms regulating the change in capsid size have been reported. Here, we investigated how a satellite from Vibrio cholerae, PLE, remodels the capsid it has been predicted to steal from the phage ICP1 (1). We identified that a PLE-encoded protein, TcaP, is both necessary and sufficient to form small capsids during ICP1 infection. Interestingly, we found that PLE is dependent on small capsids for efficient transduction of its genome, making it the first satellite to have this requirement. ICP1 isolates that escaped TcaP-mediated remodeling acquired substitutions in the coat protein, suggesting an interaction between these two proteins. With a procapsid-like-particle (PLP) assembly platform in Escherichia coli, we demonstrated that TcaP is a bona fide scaffold that regulates the assembly of small capsids. Further, we studied the structure of PLE PLPs using cryogenic electron microscopy and found that TcaP is an external scaffold, that is functionally and somewhat structurally similar to the external scaffold, Sid, encoded by the unrelated satellite P4 (2). Finally, we showed that TcaP is largely conserved across PLEs. Together, these data support a model in which TcaP directs the assembly of small capsids comprised of ICP1 coat proteins, which inhibits the complete packaging of the ICP1 genome and permits more efficient packaging of replicated PLE genomes.

Nuclease genes occupy boundaries of genetic exchange between bacteriophages.

Homing endonuclease genes (HEGs) are ubiquitous selfish elements that generate targeted double-stranded DNA breaks, facilitating the recombination of the HEG DNA sequence into the break site and contributing to the evolutionary dynamics of HEG-encoding genomes. Bacteriophages (phages) are well-documented to carry HEGs, with the paramount characterization of HEGs being focused on those encoded by coliphage T4. Recently, it has been observed that the highly sampled vibriophage, ICP1, is similarly enriched with HEGs distinct from T4’s. Here, we examined the HEGs encoded by ICP1 and diverse phages, proposing HEG-driven mechanisms that contribute to phage evolution. Relative to ICP1 and T4, we found a variable distribution of HEGs across phages, with HEGs frequently encoded proximal to or within essential genes. We identified large regions (> 10kb) of high nucleotide identity flanked by HEGs, deemed HEG islands, which we hypothesize to be mobilized by the activity of flanking HEGs. Finally, we found examples of domain swapping between phage-encoded HEGs and genes encoded by other phages and phage satellites. We anticipate that HEGs have a larger impact on the evolutionary trajectory of phages than previously appreciated and that future work investigating the role of HEGs in phage evolution will continue to highlight these observations.