Biology subjects

Torrance, E. L.

Publications and source records attributed to Torrance, E. L..

4 recordsLinked to original sources

bioRxiv2026

Temperature-Switchable Genome Editors from Extremophile-Derived Integrases

Integrases are site-specific recombinases encoded by phages and other mobile genetic elements. They mediate DNA integration, excision, and inversion between cognate attachment (att) sites. Although integrases are powerful tools for genetic engineering and synthetic biology, most systems lack intrinsic mechanisms that limit activity after expression, creating potential for unintended recombination. We hypothesized that extremophiles could provide temperature-responsive integrases because their enzymes evolved under selective pressure to operate within the thermal ranges experienced by their hosts. To test this concept, we linked integrase-att pairs from 458,683 prokaryotic genome assemblies to curated host growth-temperature metadata. This analysis revealed temperature-associated structure among integrase clusters and established a candidate pool for testing temperature-responsive recombinases. GC content in tyrosine integrase-associated attB sites showed a modest increase in higher-temperature hosts. We developed an inversion assay using a single-copy reporter plasmid and sacB counterselection to quantify integrase activity across temperatures. Thermophile derived integrases from Thermus thermophilus and Geobacillus stearothermophilus displayed hot-ON/cold-OFF activity profiles, whereas an integrase derived from the psychrotroph Pseudomonas cerasi showed cold-ON/hot-OFF activity. Together, these results establish host thermal niche as a guide for discovering intrinsically temperature switchable integrases and provide a foundation for engineering thermally controlled genome editing systems.

molecular biology↗

bioRxiv2025

Integrase-On-Demand: Bioprospecting integrases for targeted genomic insertion of genetic cargo

Integrases recognize defined sites within DNA and facilitate homologous recombination-independent transfer of genetic fragments. These innate capabilities render integrases as powerful biotechnology tools that mediate efficient site-specific integration of large genetic cargos. Given that genomes often lack the integration sites recognized by frequently utilized integrases, integrase technology has largely been restricted to genetic engineering of model organisms into which attB sites can be synthetically introduced. To enable single-step site-specific integrase-mediated genome editing in a broad spectrum of prokaryotes, we have devised the Integrase-On-Demand (IOD) method. IOD systematically identifies integrases, from bacteria and archaea, that can integrate into available attB sites in any target prokaryote. Computational results show that diverse bacteria generally have multiple potentially useable native attB/integrase pairs. We confirmed the functionality of predicted integrase and attB pairs for mediating site-specific genomic integration of heterologous DNA into the genomes of Pseudomonas putida S12 and KT2440 and Synechococcus elongatus UTEX 2973 and measured efficiency of integration using suicide constructs. By eliminating the requirement to introduce non-native attB sites into the target genome, IOD may, when suitable transformation methods exist, allow facile genome integration of large constructs in non-model and possibly non-culturable bacteria. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/676606v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1442a69org.highwire.dtl.DTLVardef@3c91b1org.highwire.dtl.DTLVardef@8131f4org.highwire.dtl.DTLVardef@1f6a206_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Homologous Recombination Shapes the Architecture and Evolution of Bacterial Genomes

Homologous recombination is a key evolutionary force that varies considerably across bacterial species. However, how the landscape of homologous recombination varies across genes and within individual genomes has only been studied in a few species. Here, we used Approximate Bayesian Computation to estimate the recombination rate along the genomes of 145 bacterial species. Our results show that homologous recombination varies greatly along bacterial genomes and shapes many aspects of genome architecture and evolution. The genomic landscape of recombination presents several key signatures: rates are highest near the origin of replication in most species, patterns of recombination generally appear symmetrical in both replichores (i.e. replicational halves of circular chromosomes) and most species have genomic hotpots of recombination. Furthermore, many closely related species share conserved landscapes of recombination across orthologs indicating that recombination landscapes are conserved over significant evolutionary distances. We show evidence that recombination drives the evolution of GC-content through increasing the effectiveness of selection and not through biased gene conversion, thereby contributing to an ongoing debate. Finally, we demonstrate that the rate of recombination varies across gene function and that many hotspots of recombination are associated with adaptive and mobile regions often encoding genes involved in pathogenicity.

evolutionary biology↗

bioRxiv2020

Unveiling Crucivirus Diversity by Mining Metagenomic Data

The discovery of cruciviruses revealed the most explicit example of a common protein homologue between DNA and RNA viruses to date. Cruciviruses are a novel group of circular Rep-encoding ssDNA (CRESS-DNA) viruses that encode capsid proteins (CPs) that are most closely related to those encoded by RNA viruses in the family Tombusviridae. The apparent chimeric nature of the two core proteins encoded by crucivirus genomes suggests horizontal gene transfer of CP genes between DNA and RNA viruses. Here, we identified and characterized 451 new crucivirus genomes and ten CP-encoding circular genetic elements through de novo assembly and mining of metagenomic data. These genomes are highly diverse, as demonstrated by sequence comparisons and phylogenetic analysis of subsets of the protein sequences they encode. Most of the variation is reflected in the replication associated protein (Rep) sequences, and much of the sequence diversity appears to be due to recombination. Our results suggest that recombination tends to occur more frequently among groups of cruciviruses with relatively similar capsid proteins, and that the exchange of Rep protein domains between cruciviruses is rarer than gene exchange. Altogether, we provide a comprehensive and descriptive characterization of cruciviruses. IMPORTANCEViruses are the most abundant biological entities on Earth. In addition to their impact on animal and plant health, viruses have important roles in ecosystem dynamics as well as in the evolution of the biosphere. Circular Rep-encoding single-stranded (CRESS) DNA viruses are ubiquitous in nature, many are agriculturally important, and are viruses that appear to have multiple origins from prokaryotic plasmids. CRESS-DNA viruses such as the cruciviruses, have homologues of capsid proteins (CPs) encoded by RNA viruses. The genetic structure of cruciviruses attests to the transfer of capsid genes between disparate groups of viruses. However, the evolutionary history of cruciviruses is still unclear. By collecting and analyzing cruciviral sequence data, we provide a deeper insight into the evolutionary intricacies of cruciviruses. Our results reveal an unexpected diversity of this virus group, with frequent recombination as an important determinant of variability.

evolutionary biology↗