Biology subjects

Gilbert, C.

Publications and source records attributed to Gilbert, C..

At least 19 recordsLinked to original sources

bioRxiv2025

A scalable transposon mutagenesis system for non-model bacteria

Transposon mutagenesis enables genome-wide interrogation of gene function with a single self-contained genetic construct. However, its application to non-model bacteria remains limited because transposition efficiency depends on multiple host factors that are difficult to predict a priori including transposase activity, antibiotic resistance marker performance, and regulatory element compatibility. Here, we present a scalable system to identify functional transposon configurations in non-model bacteria through pooled library screening. We selected 18 promoters across multiple bacterial phyla to independently drive expression of the transposase and antibiotic resistance marker, generating 324 promoter combinatorial variants for each of six antibiotic resistance markers. We developed a high-throughput, automated workflow to deliver all 1,944 mariner-based transposon variants in a single experiment and applied this to 92 non-model bacteria spanning multiple phyla. From this, we identified functional transposons for 43 strains, with high-level mutagenesis (102-104 unique insertions) in 13 species, including seven with no previously described transposon mutagenesis. We then expanded to a dual-transposase system, mariner or Tn5, and devised a single transposon insertion sequencing method for high-throughput screening of 3,888 configurations. To demonstrate the practical utility of our screening approach, we used a top-performing variant to generate a genome-wide transposon mutant library for Comamonas testosteroni KF-1, a bacterium that metabolizes plastic- and lignin-derived polymers. We assayed this C. testosteroni mutant library to identify enzymatic pathways, transporter genes, and regulators essential for the metabolism of plastics-associated monomer terephthalate and lignin-associated monomer 4-hydroxybenzoate. Together, this work establishes a scalable approach to construct and identify genetic perturbation systems in non-model bacteria, expanding our ability to systematically probe gene function across the bacterial tree of life.

synthetic biology↗

bioRxiv2025

A genetic platform for a biocementation bacterium

Sporosarcina pasteurii is the most widely studied bacterium for microbially-induced calcium carbonate precipitation (MICP), a process of intense interest for materials and construction applications. Despite two decades of investigation, S. pasteurii has remained genetically intractable, limiting our mechanistic understanding of biomineralization pathways and constraining efforts to engineer scalable solutions. Here, we present the first genetic toolkit for S. pasteurii, including a stable replicating plasmid, a conjugation-based DNA delivery protocol, engineered inducible promoters, and methods for genome modification. Using homologous recombination, we precisely deleted 5.7 kb of the genome spanning two operons encoding urease activity and demonstrated complete loss of biocementation. We also screened a library of engineered transposon constructs for activity in S. pasteurii and generated a genome-wide mutant library with >15,000 unique insertion sites. Using this library, we identified putative genes affecting ureolytic growth, revealing previously inaccessible aspects of S. pasteurii genetics. This work establishes S. pasteurii as a genetically tractable platform for rational engineering of MICP and constitutes the first genetic modification capability within the Sporosarcina genus.

synthetic biology↗

bioRxiv2025

Active learning guides automated discovery of DNA delivery via electroporation for non-model microbes

Delivery of recombinant DNA is foundational for understanding and engineering a target organism. Electroporation can be applied to any cell type, yet identification of a working protocol for new organisms remains bespoke and laborious because no systematic framework exists, and appropriate instrumentation is lacking. Here, we describe an automated high-throughput platform which uses active learning to discover electroporation protocols for non-model microbes. We first devised a 24-condition electroporation screen, based on systematic evaluation and selection of key parameters, that can be applied to any microbe. Facilitated by a custom-built fully-programmable electroporator, we successfully identified electroporation protocols for eight non-model bacteria using this screen alone. We then combined this electroporation screen with our pooled POSSUM plasmid library to simultaneously evaluate 408 experimental conditions per organism and identified both a protocol and a replicating plasmid for five non-model Proteobacteria spanning three major classes (alpha-, beta-, and gamma-). We report the first electroporation protocols for Shewanella indica, Shewanella putrefaciens 200, Shewanella putrefaciens 95, Halomonas elongata, Piscinibacter sakaiensis, and Duganella zoogloeoides, as well as multiple alternative protocols for Shewanella amazonensis, Shewanella oneidensis, Azospirillum brasilense, Cupriavidus necator, Pseudomonas alcaliphila, and Escherichia coli. Finally, we developed an active learning pipeline to guide the selection of parameters based on gathered experimental data. Using our robotic platform, we iteratively tested 538 conditions over three iterations to improve electroporation for the emerging industrial chassis C. necator, achieving 8.6-fold higher transformation efficiency than state of the art. This work establishes a discovery platform for DNA delivery to diverse and recalcitrant microbes that can be extended broadly to non-model organisms in our biosphere.

microbiology↗

bioRxiv2025

Functional genomics in a microbe that degrades and metabolizes PET plastic

Piscinibacter sakaiensis (formerly Ideonella sakaiensis) was the first bacterial species known to both completely degrade and assimilate polyethylene terephthalate (PET). However, the absence of efficient genetic tools has limited direct engineering of this organism, forcing most efforts to rely on heterologous expression of PET-degrading enzymes in model hosts. Here, we establish foundational genetic tools to engineer P. sakaiensis. We identify a functional plasmid origin of replication, multiple new selectable markers, and a transposon system for the strain. We use these tools to construct a genome-wide, barcoded transposon mutant library for pooled high-throughput functional screens. We apply this mutant library to growth on PET and identify metabolic and physiological genes that impact PET biodegradation. We also use this library to reveal mutants with improved DNA uptake for genome engineering. Together, these advances provide a platform for functional genomics in P. sakaiensis and positions this naturally evolved plastic-degrading bacterium as an engineerable chassis for synthetic biology and sustainable materials research.

microbiology↗

bioRxiv2025

A reference set of functional plasmids for Vibrio natriegens

New microbial hosts with superior phenotypes, such as fast growth, are attractive for research and biotechnology, but often lack systematic evaluation of functional genetic parts. A reference set of working plasmids would increase reproducibility and encourage use of these hosts. Here, we use the POSSUM toolkit, a collection of 23 origins-of-replication and 6 antibiotic markers, to identify functional genetic parts for strains of Vibrio natriegens. We applied this to the wild-type strain ATCC 14048 and an engineered variant NBx CyClone, evaluating 414 combinations of origins of replication and antibiotic selection conditions. We show that both strains support five replicons (pNG2, pSa, pSC101ts, p15A, and RSF1010) with NBx CyClone supporting an extra replicon RK2. The assay can be performed in under a week and is compatible with multiple DNA delivery methods. This work demonstrates the feasibility of rapidly establishing reference information to accelerate the adoption of new microbial hosts.

synthetic biology↗

bioRxiv2025

Knowledge, Attitudes and Practices on Antimicrobial Resistance Among Practising Veterinary and Para-veterinary Professionals in Zambia

In this study, we aimed to assess the knowledge, attitudes, and practices (KAP) of veterinary and para-veterinary professionals concerning antimicrobial resistance (AMR), in order to identify key drivers of inappropriate antimicrobial use and gaps in current stewardship efforts. Data was collected from a cross-sectional survey of 144 veterinary and para-veterinary professionals (n=144) using an online structured self-administered questionnaire in ZOHO software. We analyzed data using descriptive statistics and logistic regression in IBM SPSS Statistics Version 25. The findings revealed a high foundational knowledge of AMR, with 66.7% correctly defining AMR. The majority of respondents (82%) acknowledged that overuse of antimicrobials reduces their effectiveness and expressed strong support for mitigation efforts, with 95.8% endorsing farmer education as a key strategy. However, significant gaps in practice persisted: 75% of animal owners frequently initiated antibiotic use without supervision; professionals largely relied on empirical treatment (62.5%); and only 20.8% of professionals routinely performed antimicrobial susceptibility testing (AST), due to barriers like cost and laboratory accessibility. Logistic regression analysis revealed no significant associations between socio-demographic variables and levels of AMR knowledge or attitudes. This suggests that barriers to responsible antimicrobial use may be more structural than individual. We conclude that while Zambian practicing veterinary and para-veterinary professionals possess strong AMR awareness and positive attitudes, risky practices do persist. We recommend strengthening AMR stewardship with a holistic One Health approach by improving veterinary services to promote more appropriate and effective antimicrobial use. Author SummaryAntibiotics are essential for treating sick animals, but their misuse can lead to antimicrobial resistance, where bacteria no longer respond to the drugs meant to kill them. This problem threatens both animal and human health. In Zambia, veterinary and para-veterinary professionals are actively involved in administering treatments and supporting decisions related to antibiotic use in livestock and pets. We wanted to understand what these professionals know, believe, and do when it comes to using antibiotics responsibly. To find out, we surveyed 144 veterinary and para-veterinary professionals from across the country. We learned that most of them were aware that misuse of antibiotics can make treatments less effective and that drug-resistant bacteria can spread between animals and people. Many also supported educating farmers and working with human health experts to tackle the issue. However, we also discovered that antibiotics were often used in animals without proper testing or diagnosis, mainly because of limited laboratory access and cost concerns. Our findings show that while knowledge and attitudes are strong, actual practices need improvement. We believe improved access to diagnostic services, more explicit rules, and ongoing training can help veterinary and veterinary paraprofessionals protect both animal and public health from the growing threat of antibiotic resistance.

scientific communication and education↗

bioRxiv2025

Deep learning-based photo-identification for non-invasive monitoring of animal populations: Application to penguins and tortoises

We describe DL-ID, a deep learning-based framework for the individual identification of animals from images with higher accuracy and characteristics that make it more robust in challenging conditions, such as field studies, than widely used algorithms. This is demonstrated through testing with small datasets involving two species that exhibit distinctive morphological features: Humboldt penguins (Spheniscus humboldti Meyen, 1834) and Hermanns tortoises (Testudo hermanni Gmelin, 1789). We define a confidence index based on entropy within the model that significantly improves handling of the open set recognition problem, offering a new way to discriminate unknown individuals. DL-IDs accuracy of 87%, even with as few as four images per class, contrasts with the usual practice in deep learning of building large datasets to assess performance. The model outperforms traditional photo-identification methods like Wild-ID and I3S Pattern, offering a significant advancement for research and conservation efforts. Its efficiency and adaptability indicate its potential in real-time monitoring, opening new possibilities for wildlife conservation. Instead of designing a complex new deep learning model, we focused on adapting existing methods to address a relevant ecological problem. Our approach effectively tackles issues like limited training data and recognizing new individuals in field studies. Notably, it performs well even with small datasets, making it particularly useful for data-limited ecological research. This makes our approach a valuable step forward in applying deep learning to ecological studies.

ecology↗

bioRxiv2025

No Evidence of Direct Activation of Human Neutrophil Responses by Multivalent Prefusion Trimeric SARS-CoV-2 Spike Protein ex vivo

The SARS-CoV-2 Spike (S) protein is essential for viral entry and serves as the primary immunogen in most COVID-19 vaccines. While its role in adaptive immunity is well defined, its potential to contribute directly to innate immune activation and remains incompletely understood. Neutrophils, in particular, are prominent effectors in COVID-19 severity, yet how they respond directly to the S protein presented in a multivalent format is unclear. Here, we investigated whether the S protein can directly activate human neutrophils ex vivo using two biologically relevant models: nanoparticles displaying multivalent stabilized prefusion trimeric S glycoprotein, and purified {beta}-propiolactone-inactivated SARS-CoV-2 virions. Neutrophils were exposed to nanoparticles or inactivated virus, either alone or pre-coated with monoclonal or polyclonal anti-S antibodies. Nanoparticles displaying Respiratory Syncytial Virus (RSV) Fusion (F) protein and purified {beta}-propiolactone-inactivated RSV served as comparators. Across all models and conditions tested, the S protein did not induce significant neutrophil responses. No consistent effects were observed on cell viability, surface marker expression, reactive oxygen species production, neutrophil extracellular trap formation, cytokine release, or inflammatory gene expression--even in the presence of anti-S antibodies mimicking immune complexes. Results with F-nanoparticles and inactivated RSV were similarly modest. These findings indicate that the trimeric prefusion S protein, whether displayed multivalently on nanoparticles or in the context of inactivated viral particles, is insufficient to trigger robust neutrophil activation. This work provides insight into the innate immune profile of the S protein and suggests that its use in vaccine platforms is unlikely to directly provoke neutrophil-mediated inflammatory responses.

immunology↗

bioRxiv2025

Characterization of HIV-1 particles co-purified with three extracellular vesicle subtypes from the Raji CD4 DCIR cell line, a hybrid model of CD4 T cells and dendritic cells

BackgroundHIV-1 proteins and RNA are packaged into extracellular vesicles (EVs) through interaction with the multivesicular endosomes of the EV biogenesis machinery. This interaction also allows functional or abortive viruses to exit the cells as exosomes, microvesicles or apoptotic vesicles. HIV-1 viral particles and EVs share significant similarities in size, composition, and molecular cargo, making their separation challenging. Current HIV-1 purification methods neglect the effects of EVs on virus infectivity, which could influence experiment outcomes. Here, we co-characterized HIV-1 particles co-purified with exosomes, microvesicles and apoptotic vesicles to determine their impact on HIV-1 infection. MethodsThe HIV-infected Raji CD4 DCIR cells supernatants were harvested 2 and 8 days after infection. The 2-day supernatant was treated with proteinase K to discard viral protein and HIV-1 RNA associated with proteins outside the EVs. The supernatants were fractionated into three pellets by differential centrifugation. The 3K pellet contained the largest EVs, such as apoptotic vesicles. The 17K and 100K pellets were respectively associated with microvesicles and exosomes. EVs and viral particles were co-characterized for their host and viral contents and the pellets obtained after 8 days post-infection were tested for infectivity. ResultsProteinase K treatment notably lowered HIV-1 RNA concentration in the EV pellet and did not affect viral p24 capsid protein concentration. The p24 protein was mostly found in the 17K pellet and HIV-1 RNA was the most abundant in the 100K pellet for both 2- and 8-day productions. Nevertheless, the 3K pellet had the highest infectivity when cells were infected with an equal quantity of virus (measured by p24) from each pellet. ConclusionProductively infected cells released functional viruses in the three EV subtypes, each exerting a distinct effect on virus infectivity. For future experiments, the presence of EVs in viral preparations should be taken into account, as they influence the progression of HIV-1 infection. HighlightsO_LIHIV-1 RNA and p24 capsid protein are packaged within apoptotic vesicles, microvesicles and exosomes released by Raji CD4 DCIR cells. C_LIO_LIInfectious particles co-precipitated with the 3K pellet from Raji CD4 DCIR cells are more infectious than other viruses associated with other EV pellets. C_LI

cell biology↗

bioRxiv2024

Phylogenetic relatedness rather than aquatic habitat fosters horizontal transfer of transposable elements in animals

Horizontal transfer of transposable elements (HTT) is an important driver of genome evolution, yet the factors conditioning this phenomenon remain poorly characterized. Here, we screened 247 animal genomes from four phyla (annelids, arthropods, mollusks, chordates), spanning 19 independent transitions between aquatic and terrestrial lifestyles, to evaluate the suspected positive effects of aquatic habitat and of phylogenetic relatedness on HTT. Among the 5,952 independent HTT events recovered, the vast majority (>85%) involve DNA transposons, of which Mariner-like and hAT-like elements have the highest rates of horizontal transfer, and of intra-genomic amplification. Using a novel approach that circumvents putative biases linked to phylogenetic inertia and taxon sampling, we found that HTT rates positively correlate with similarity in habitat type but were not significantly higher in aquatic than in terrestrial animals. However, modelling the number of HTT events as a function of divergence time in a Bayesian framework revealed a clear positive effect of phylogenetic relatedness on HTT rates in most of the animal species studied (162 out of 247). The effect is very pronounced: a typical species is expected to show 10 times more transfers with a species it diverged from 125 million years (My) ago than with a species it diverged from 375 My ago. Overall, our study underscores the pervasiveness of HTT throughout animals and the impact of evolutionary relatedness on its dynamics. Significance statementGenetic material can be transmitted between organisms through other means than reproduction, in a process called horizontal transfer. The mechanisms and factors underlying this phenomenon in animals remain unclear, although it often involves transposable elements (TEs). TEs are DNA segments capable of jumping within genomes, but also occasionally between individuals. Here, we show evidence for nearly 6,000 transfers of TEs among animals, based on genomic comparisons among 247 species of annelids, arthropods, chordates and mollusks. Contrarily to expectations, we found no excess in the rates of transfers in aquatic versus terrestrial animals. By contrast, most analyzed species appeared engaged in many more horizontal transfers with close than with distant relatives, highlighting the strong impact of phylogenetic relatedness on horizontal transfers of TEs.

evolutionary biology↗

bioRxiv2024

Strong evidence for multiple horizontal transfers of immune genes between teleost fishes

Horizontal gene transfer (HGT) is less frequent in eukaryotes than in prokaryotes, yet can have strong functional implications and was proposed as causal factor for major adaptations in several eukaryotic lineages. Most cases of eukaryote HGT reported to date are inter-domain transfers and only few studies have investigated eukaryote-to-eukaryote HGTs. Furthermore, the extent to which HGT has contributed to the gene repertoire of some lineages such as vertebrates remains unclear. Here, we performed a large-scale survey of HGT among 242 species of ray-finned fishes. We found strong evidence supporting 18 teleost-to-teleost HGT events that involve 17 different genes in 11 teleost fish orders. The genes involved in these transfers show lower synonymous divergence than expected under vertical transmission, their phylogeny is inconsistent with that of teleost fishes, and high nucleotide similarity on these genes between donor and recipient lineages extends to non-coding regions. The distribution of HGT events in the teleost tree is heterogenous, with eight of the 18 transfers occurring between the same two orders (Osmeriformes and Clupeiformes). Besides the previously reported transfer of an antifreeze protein, most transferred genes play roles in immunity, suggesting that such genes are more likely than others to confer strong selective advantage to the recipient species. Overall, our work shows that teleost-to-teleost HGT has occurred on multiple occasions and it will be worth further quantifying these transfers and evaluating their impact on teleost evolution as more genomes are being sequenced.

evolutionary biology↗

bioRxiv2024

Exploring the Relationship Between Extracellular Vesicles, the Dendritic Cell Immunoreceptor and microRNA-155 in an In Vivo Model of HIV-1 Infection to Understand the Disease and Develop New Treatments

HIV-1 infection induces persistent immune system activation despite antiretroviral therapy. New immunomodulatory targets might be required to restore immune competence. The dendritic cells immunoreceptor (DCIR) can bind HIV-1 and regulate immune functions and extracellular vesicles (EVs) production. EVs have emerged as biomarkers and a non-invasive tool to monitor HIV-1 progression. In people living with HIV-1, an increase in the size and abundance of EVs is associated with a decline in the CD4/CD8 T cells ratio, a key marker of immune dysfunction. Analysis of host nucleic acids within EVs has revealed an enrichment of microRNA-155 (miR-155) during HIV-1 infection. Experiments have demonstrated that miR-155-rich EVs enhance HIV-1 infection in vitro. A humanized NSG-mice model was established to assess the in vivo impact of miR-155-rich EVs. Co-production of virus with miR-155-rich EVs heightened the viral load and lowered the CD4/CD8 ratio in the mice. Upon euthanasia, EVs were isolated from plasma for size and quantity assessment. Consistent with findings in individuals with HIV-1, increased EVs size and abundance were inversely correlated with the CD4/CD8 ratio. Next, by using the more closely related physiological virus co-product with EV-miR-155, we tested a DCIR inhibitor to limit infection and immune damage in a humanized mouse model. DCIR inhibition reduced infection and partially restored immune functions. Finally, viral particles and various EV subtypes can convey HIV-1 RNA. HIV-1 RNA was predominantly associated with large EVs (200-1000nm) rather than small EVs (50-200nm). Viral loads in large EVs strongly correlated with blood and tissue markers of immune activation. The humanized mice model has proven its applicability to studying the roles of EVs on HIV-1 infection and investigating the impact of DCIR inhibition. Author SummaryDespite more than 40 years of research, HIV remains a threat to public health around the world. People living with HIV are efficiently treated with antiretroviral therapy, but damage to the immune system persists and the causes remain unknown. Extracellular vesicles allow material, such as microRNA, to transfer between cells. Here, we evaluated the impact of one microRNA, microRNA-155, transported by extracellular vesicles, on HIV infection. Mice were grafted with a human immune system to allow infection by HIV. We showed that extracellular vesicles carrying microRNA-155 amplified mice infection. Extracellular vesicles also reflect the state of their cell of origin. Their analysis can reveal biomarkers to monitor HIV infection. Thus, HIV viral load was quantified in purified extracellular vesicles. We found that the measurement of HIV viral load in purified EVs is a more precise biomarker of disease progression than the traditional plasma viral load. Additionally, potential treatments like DCIR inhibitors improve our ability to manage HIV-1 by restoring the CD4/CD8 ratio, a critical element of the infection process. Overall, our study highlighted the importance of extracellular vesicle cargo in a humanized mouse model of HIV-1 infection, as well as the potential of targeting DCIR to restore the immune response. HighlightsO_LIMicroRNA-155 promotes HIV-1 infection of humanized NSG mice C_LIO_LIAbundance and size of total plasmatic EVs are biomarkers of immune dysfunction associated with HIV-1 infection C_LIO_LIDCIR inhibition limits HIV-1 infection of humanized NSG mice and attenuates immune impairment C_LIO_LIHIV-1 RNA enrichment in large EVs was associated with biomarkers of immune activation and dysfunction C_LI

immunology↗

bioRxiv2024

Viral overexpression of human alpha-synuclein in mouse substantia nigra dopamine neurons results in hyperdopaminergia but no neurodegeneration

Loss of select neuronal populations such as midbrain dopamine (DA) neurons is a pathological hallmark of Parkinsons disease (PD). The small neuronal protein -synuclein has been related both genetically and neuropathologically to PD, yet how it contributes to selective vulnerability remains elusive. Here, we describe the generation of a novel adeno-associated viral vector (AAV) for Cre-dependent overexpression of wild-type human -synuclein. Our strategy allows us to restrict -synuclein to select neuronal populations and hence investigate the cell-autonomous effects of elevated -synuclein in genetically-defined cell types. Since DA neurons in the substantia nigra pars compacta (SNc) are particularly vulnerable in PD, we investigated in more detail the effects of increased -synuclein in these cells. AAV-mediated overexpression of wildtype human -synuclein in SNc DA neurons increased the levels of -synuclein within these cells and augmented phosphorylation of -synuclein at serine-129, which is considered a pathological feature of PD and other synucleinopathies. However, despite abundant -synuclein overexpression and hyperphosphorylation we did not observe any DA neurodegeneration up to 90 days post virus infusion. In contrast, we noticed that overexpression of -synuclein resulted in increased locomotor activity and elevated striatal DA levels suggesting that -synuclein enhanced dopaminergic activity. We therefore conclude that cell-autonomous effects of elevated -synuclein are not sufficient to trigger acute DA neurodegeneration.

neuroscience↗

bioRxiv2024

Design and construction towards a pan-microbial toolkit

Establishing genetic tractability in non-model microbes requires identifying genetic parts that function in a target host. However, the paucity and purported narrow host range of available parts means that successful identification is governed by serendipity. Instead, a more comprehensive and scalable process would be desirable. Here, we describe the design principles for a pan-microbial genetic toolkit in which phylogenetically-diverse parts can be assembled and tested for function in microbes using high-throughput readouts. The architecture is based on Golden Gate Assembly, which simplifies the addition of parts and the construction of combinatorial libraries. We used this framework to develop two modules: first, the POSSUM (Plasmid Origins and Selection MarkerS for Undomesticated Microbes) module for identification of replicating plasmids in non-model microbes which includes 29 plasmid origin of replication sequences, 23 selection markers, and 30 unique DNA sequences for tracking by sequencing; second, the MACKEREL (Modular, NGS-trACKable ExpRession ELement) module, for identification of functional gene expression cassettes which includes 426 bacterial promoter-RBS sequences driving fluorescent reporter expression, trackable by flow cytometry. We demonstrate the use of these libraries to screen for functional promoter-RBS variants in 6 non-model microbes. Continued efforts to expand this pan-microbial toolbox will accelerate efforts to improve genetic tractability and guide research across the tree of life.

synthetic biology↗

bioRxiv2024

Systematic annotation of Helitron-like elements in eukaryote genomes using HELIANO

Helitron-like elements (HLEs) are widespread eukaryotic DNA transposons employing a rolling-circle transposition mechanism. Despite their prevalence in fungi, animals, and plant genomes, identifying Helitrons remains challenging. We introduce HELIANO, a software for annotating and classifying autonomous and non-autonomous Helitron and Helentron sequences from whole genomes. HELIANO outperforms existing tools in speed and accuracy, demonstrated through benchmarking and its application to complex genomes (Xenopus tropicalis, Xenopus laevis, Oryza sativa), revealing numerous newly identified Helitrons and Helentrons. In a comprehensive analysis of 404 eukaryote genomes, we found HLEs widely distributed across phyla, with exceptions in specific taxa. Helentrons were identified in numerous land plant species, and 20 protein domains were discovered integrated within specific autonomous HLE families. A global phylogenetic analysis confirmed the classification into main clades Helentron and Helitron, revealing nine subgroups, some enriched in particular taxa. The future use of HELIANO will contribute to the global analysis of TEs across genomes and enhance our understanding of this transposon superfamily.

evolutionary biology↗

bioRxiv2023

Architecture and evolutionary conservation of Xenopus tropicalis osteoblast-specific regulatory regions shed light on bone diseases and early skeletal evolution

Understanding the genetic mechanisms underpinning the differentiation of osteoblasts, the bone producing cells, has far reaching implications for skeletal diseases and evolution. To this end, it is crucial to characterize osteoblastic regulatory landscape in a diverse array of distantly-related vertebrate species. By comparing of the ATAC-seq profile of Xenopus tropicalis (Xt) osteoblasts to liver, heart and lung control tissues, we identified 524 promoters and 6,750 distal regions whose chromatin is specifically open in osteoblasts. Nucleotide composition, Gene Ontology, and RNA-Seq confirmed that the identified elements correspond to bona fide osteogenic transcriptional enhancers, and TFBS enrichment revealed a well-conserved regulatory logic with mammals. Amongst the 357 Xt osteoblast-specific enhancers aligning to homologous human loci, 127 map to regions annotated as enhancers. Phenotype predictions based on the genes neighbouring these conserved enhancers are tightly related to impaired skeletal development. In addition, six conserved enhancers are located at loci associated to craniosynostosis (mx2, tcf12), osteopoikilosis (lemd3), osteopenia (gorab), skeletal dysplasia (flnb) and craniofacial abnormalities (gpc4). From an evolutionary perspective, the elephant shark genome aligns to 53 Xt osteoblast-specific enhancers that are also conserved and annotated as enhancers in humans, revealing an ancestral osteogenic role for the ATOH8, IRX3, NFAT, NFIB and MEF2C transcription factors, as well as for the FGF, IHH and BMP/TGFb signalling pathways. As the absence of bone in sharks is a derived feature, we propose that, in this lineage, the osteogenic regulatory network has been maintained for its function in odontoblasts. Our data argues in favour of a common origin for dentine and bone, and provides a glimpse into the key regulatory elements and upstream activators that drove the formation of an ancient type of mineralized tissue in the vertebrates that inhabited the oceans more than 460 million years ago. Author SummaryDuring animal embryogenesis, distinct type of tissues are formed and assembled, resulting in an integrated, functional organism. During this process, cells must make important decisions, which largely rely on an accurate use of their genetic material. Here, we have studied how the genome "knows" that it must participate to the formation of the bone tissue in a frog animal model. We therefore identified important genomic regions that are involved in driving the expression of genes involved in the formation of a mineralized skeleton. On the one hand, we show that some of these regions are also present in humans, and, therefore, skeletal pathologies could be studied in the frog model at a genetic level. On the other hand, we also identify regions that are present in the genome of a shark, which allows us to propose an evolutionary framework for the early evolutionary origin of the vertebrate skeleton.

genomics↗

bioRxiv2023

Enamel proteins reveal biological sex and genetic variability within southern African Paranthropus

The evolutionary relationships among extinct African hominin taxa are highly debated and largely unresolved, due in part to a lack of molecular data. Even within taxa, it is not always clear, based on morphology alone, whether ranges of variation are due to sexual dimorphism versus potentially undescribed taxonomic diversity. For Paranthropus robustus, a Pleistocene hominin found only in South Africa, both phylogenetic relationships to other taxa 1,2 and the nature of intraspecific variation 3-6 are still disputed. Here we report the mass spectrometric (MS) sequencing of enamel proteomes from four ca. 2 million year (Ma) old dental specimens attributed morphologically to P. robustus, from the site of Swartkrans. The identification of AMELY-specific peptides and semi-quantitative MS data analysis enabled us to determine the biological sex of all the specimens. Our combined molecular and morphometric data also provide compelling evidence of a significant degree of variation within southern African Paranthropus, as previously suggested based on morphology alone 6. Finally, the molecular data also confirm the taxonomic placement of Paranthropus within the hominin clade. This study demonstrates the feasibility of recovering informative Early Pleistocene hominin enamel proteins from Africa. Crucially, it also shows how the analysis of these proteins can contribute to understanding whether hominin morphological variation is due to sexual dimorphism or to taxonomic differences. We anticipate that this approach can be widely applied to geologically-comparable sites within South Africa, and possibly more broadly across the continent.

evolutionary biology↗

bioRxiv2023

A scalable framework for high-throughput identification of functional origins of replication in non-model bacteria

Microbial genetic manipulation requires access to engineerable plasmids that can be programmed to perturb genes, pathways and genomes. The extensive repertoire of plasmids available for model microbes, such as Escherichia coli, has facilitated fundamental biology studies and synthetic biology applications. However, the scarcity of plasmids for non-model microbes hinders efforts to broaden our biological knowledge and constrains the development of biotechnological solutions. In this study, we introduce a molecular toolkit and multiplexed screen to evaluate functional plasmids in non-model microbes. We constructed a collection of genetic parts consisting of 22 origins of replication (ORIs), 20 antibiotic selectable markers, and 30 molecular barcodes, which can be assembled combinatorially to create a library of plasmids trackable by next-generation DNA sequencing. We demonstrate our approach by delivering a pooled library of 22 ORIs to 12 bacterial species including extremophiles, electroactive bacteria and bioproduction strains. We report, for the first time, DNA delivery by conjugation and functional ORIs for Halomonas alkaliphila, Halomonas neptunia, and Shewanella electrodiphila. Furthermore, we expand the list of functional ORIs for Duganella zoogloeoides, Pseudomonas alcaliphila, Shewanella oneidensis and Shewanella putrefaciens. This screen provides a scalable high-throughput system to rapidly build and identify functional plasmids to establish genetic tractability in non-model microbes.

synthetic biology↗