Biology subjects

Morgan, R. D.

Publications and source records attributed to Morgan, R. D..

9 recordsLinked to original sources

bioRxiv2026

A virion protein of an archaeal virus inhibits a Type V BREX defense system in Haloarcula hispanica

Bacteriophage Exclusion (BREX) systems are a diverse family of bacterial and archaeal defense mechanisms defined by two conserved genes encoding a putative alkaline phosphatase BrxZ (PglZ) and an ATPase BrxC (PglY). Here, we characterize a Type V BREX system from the archaeon Haloarcula hispanica. Similar to bacterial Type I BREX systems, host-virus discrimination relies on methylation of specific non-palindromic DNA motifs. Notably, the H. hispanica BREX locus encodes two methyltransferases, BrxX1 and BrxX2, which independently target distinct motifs (GTAYCCG and GACCCC). The system protects H. hispanica against three of seven tested archaeal viruses. However, two related viruses, SH1 and HHIV-2, despite encoding multiple BREX target sites, escape BREX-mediated defense. We demonstrate that a large virion protein, VP1, encoded by these viruses inhibits the BREX system, as mutant viruses carrying partial deletions of VP1 lose resistance to the host defense. Together, these findings provide the first characterization of a Type V BREX system and demonstrate that archaeal viruses can counteract BREX through virion-associated anti-defense proteins.

molecular biology↗

bioRxiv2025

Transcriptional Circuitry in HGSOC: A Dynamic Three-State Model Informed by a Living Biobank of Purified Tumour Fractions

High-grade serous ovarian cancer (HGSOC) is a heterogeneous disease, but efforts to define transcriptional subtypes using bulk RNA sequencing have been confounded by the presence of non-malignant cells. As a result, it remains unclear whether tumour-cell-intrinsic states exist, and whether these represent stable disease subtypes or are dynamically remodelled during disease progression and treatment. Here, we address this question using a living biobank of patient-derived ovarian cancer models (OCMs) cultured as purified tumour-cell populations under uniform conditions. RNA sequencing followed by unsupervised non-negative matrix factorisation (NMF) revealed a robust, hierarchical architecture comprising three core tumour-cell-intrinsic subtypes: the Alpha cluster, marked by cell-cycle deregulation and E2F-driven replication stress; the Beta cluster, defined by tumour-cell-intrinsic immune mimicry and inflammatory signalling; and the Gamma cluster, characterised by epithelial identity, extracellular matrix engagement, and metabolic adaptation. At higher clustering resolution, a fourth cluster, Delta, emerged as a Gamma sub-lineage distinguished by a vesicle-oriented, neuronal-like secretory programme. By projecting cluster labels onto a subset of matched longitudinal OCMs using non-negative least squares, we show that while some tumours retain stable subtype identities, others display transcriptional plasticity, including transitions from epithelial-like Gamma states to more proliferative or secretory phenotypes. Together, these findings define the core architecture and dynamic potential of tumour-cell-intrinsic transcriptional states within HGSOC, thereby bridging legacy bulk classifications with emerging single-cell insights, establishing a framework for more precise patient stratification.

cancer biology↗

bioRxiv2024

Structure and rational engineering of the PglX methyltransferase and specificity factor for BREX phage defence

AO_SCPLOWBSTRACTC_SCPLOWBacteria have evolved a broad range of systems that provide defence against their viral predators, bacteriophages. Bacteriophage Exclusion (BREX) systems recognize and methylate 6 bp non-palindromic motifs within the host genome, and prevent replication of non-methylated phage DNA that encodes these same motifs. How BREX recognizes cognate motifs has not been fully understood. We have characterised BREX from pathogenic Salmonella and generated the first X-ray crystallographic structures of the conserved BREX protein, PglX. The PglX N-terminal domain encodes the methyltransferase, whereas the C-terminal domain is for motif recognition. We also present the structure of PglX bound to the phage-derived DNA mimic, Ocr, an inhibitor of BREX activity. Our analyses propose modes for DNA-binding by PglX and indicate that larger BREX complexes are required for methyltransferase activity and defence. Through rational engineering of PglX, we broadened both the range of phages targeted, and the host motif sequences that are methylated by BREX. Our data demonstrate that PglX is the sole specificity factor for BREX activity, providing motif recognition for both phage defence and host methylation.

microbiology↗

bioRxiv2023

Genomes of the human filarial parasites Mansonella perstans and Mansonella ozzardi

The filarial parasites Mansonella ozzardi and Mansonella perstans, causative agents of mansonellosis, infect hundreds of millions of people worldwide, yet remain among the most understudied of the human filarial pathogens. M. ozzardi is highly prevalent in Latin American countries and Caribbean Islands, while M. perstans is predominantly found in sub-Saharan Africa as well as in a few areas in South America. In addition to the differences in their geographical distribution, the two parasites are transmitted by different insect vectors, as well as exhibit differences in their responses to commonly used anthelminthic drugs. The lack of genome information has hindered investigations into the biology and evolution of Mansonella parasites and understanding the molecular basis of the clinical differences between species. In the current study, high quality genomes of two independent clinical isolates of M. perstans from Cameroon and two M. ozzardi isolates one from Brazil and one from Venezuela are reported. The genomes are approximately 76 Mb in size, encode about 10,000 genes each, and are largely complete based on BUSCO scores of about 90%, similar to other completed filarial genomes. These sequences represent the first genomes from Mansonella parasites and enabled a comparative genomic analysis of the similarities and differences between Mansonella and other filarial parasites. Horizontal DNA transfers (HDT) from mitochondria (nuMTs) as well as transfers from genomes of endosymbiotic Wolbachia bacteria (nuWTs) to the host nuclear genome were identified and analyzed. Sequence comparisons and phylogenetic analysis of known targets of anti-filarial drugs diethylcarbamazine (DEC), ivermectin and mebendazole revealed that all known target genes were present in both species, except for the DEC target encoded by gon-2 gene, which is fragmented in genome assemblies from both M. ozzardi isolates. These new reference genome sequences will provide a valuable resource for further studies on biology, symbiosis, evolution and drug discovery.

genomics↗

bioRxiv2022

Assembling highly repetitive Xanthomonas TALomes using Oxford Nanopore sequencing

Most plant-pathogenic Xanthomonas bacteria harbor transcription activator-like effector (TALE) genes, which function as transcriptional activators of host plant genes and support infection. The entire repertoire of up to 29 TALE genes of a Xanthomonas strain is also referred to as TALome. The DNA-binding domain of TALEs is comprised of highly conserved repeats and TALE genes often occur in gene clusters, which precludes the assembly of TALE-carrying Xanthomonas genomes based on standard sequencing approaches. Here, we report the successful assembly of the 5 Mbp genomes of five Xanthomonas strains from Oxford Nanopore Technologies (ONT) sequencing data. For one of these strains, Xanthomonas oryzae pv. oryzae (Xoo) PXO35, we illustrate why Illumina short reads and longer PacBio reads are insufficient to fully resolve the genome. While ONT reads are perfectly suited to yield highly contiguous genomes, they suffer from a specific error profile within homopolymers. To still yield complete and correct TALomes from ONT assemblies, we present a computational correction pipeline specifically tailored to TALE genes, which yields at least comparable accuracy as Illumina-based polishing. We further systematically assess the ONT-based pipeline for its multiplexing capacity and find that, combined with computational correction, the complete TALome of Xoo PXO35 could have been reconstructed from less than 20,000 ONT reads. Our results indicate that multiplexed ONT sequencing combined with a computational correction of TALE genes constitutes a highly capable tool for characterizing the TALomes of huge collections of Xanthomonas strains in the future.

bioinformatics↗

bioRxiv2021

A widespread family of WYL-domain transcriptional regulators co-localises with diverse phage defence systems and islands

AO_SCPLOWBSTRACTC_SCPLOWBacteria are under constant assault by bacteriophages and other mobile genetic elements. As a result, bacteria have evolved a multitude of systems that protect from attack. Genes encoding bacterial defence mechanisms can be clustered into "defence islands", providing a potentially synergistic level of protection against a wider range of assailants. However, there is a comparative paucity of information on how expression of these defence systems is controlled. Here, we functionally characterise a transcriptional regulator, BrxR, encoded within a recently described phage defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a combination of reporters and electrophoretic mobility shift assays, we discovered that BrxR acts as a repressor. We present the structure of BrxR to 2.15 [A], the first structure of this family of transcription factors, and pinpoint a likely binding site for ligands within the WYL-domain. Bioinformatic analyses demonstrated that BrxR homologues are widespread amongst bacteria. About half (48%) of identified BrxR homologues were co-localised with a diverse array of known phage defence systems, either alone or clustered into defence islands. BrxR is a novel regulator that reveals a common mechanism for controlling the expression of the bacterial phage defence arsenal.

microbiology↗

bioRxiv2021

Identification and characterization of BrxR as a regulatory gene in the BREX phage restriction system

Bacteriophage exclusion ( BREX) phage restriction systems are found in a wide range of bacteria. Various BREX systems encode unique combinations of proteins that usually include a site-specific methyltransferase; none appear to contain a nuclease. Here we describe the identification and characterization of a Type I BREX system from Acinetobacter and the effect of deleting each BREX ORF on growth, methylation, and restriction. We identified a previously uncharacterized gene in the BREX operon that is dispensable for methylation but involved in restriction. Biochemical and crystallographic analyses of this factor, which we term BrxR ( BREX Regulator), demonstrate that it forms a homodimer and specifically binds a DNA target site upstream of its transcription start site. Deletion of the BrxR gene causes cell toxicity, reduces restriction, and significantly increases the expression of BrxC. In contrast, the introduction of a premature stop codon into the BrxR gene, or a point mutation blocking its DNA binding ability, has little effect on restriction, implying that the BrxR coding sequence and BrxR protein play independent functional roles. We speculate that the BrxR coding sequence is involved in cis regulation of anti-phage activity, while the BrxR protein plays an additional regulatory role, perhaps during horizontal transfer.

molecular biology↗

bioRxiv2021

Genome archeology of two laboratory Salmonella enterica enterica sv Typhimurium

The Salmonella research community has used strains and bacteriophages over decades, exchanging useful new isolates among laboratories for study of cell surface antigens, metabolic pathways and restriction-modification studies. Here we present the sequences of two laboratory Salmonella strains (STK005, an isolate of LB5000; and its descendant ER3625). In the ancestry of LB5000, segments of ~15 and ~42 kb were introduced from Salmonella enterica sv Abony 103 into Salmonella enterica sv Typhimurium LT2, forming strain SD14; this strain is thus a hybrid of S. enterica isolates. Strains in the SD14 lineage were used to define flagellar antigens from the 1950s to the 1970s, and to define three restriction-modification systems from the 1960s to the 1980s. LB5000 was also used as host in phage typing systems used by epidemiologists. In the age of cheaper and easier sequencing, this resource will provide access to the sequence that underlies the extensive literature.

genomics↗

bioRxiv2021

Coordination of phage genome degradation versus host genome protection by a bifunctional restriction-modification enzyme visualized by CryoEM

Restriction enzymes that combine DNA methylation and cleavage activities into a single polypeptide or protein assemblage and that modify just one DNA strand for host protection are capable of more efficient adaptation towards novel target sites. However, they must solve the problem of discrimination between newly replicated and unmodified host sites (needing methylation) and invasive foreign site (needing to lead to cleavage). One solution to this problem might be that the activity that occurs at any given site is dictated by the oligomeric state of the bound enzyme. Methylation requires just a single bound site and is relatively slow, while cleavage requires that multiple unmethylated target sites (often found in incoming, foreign DNA) be brought together into an enzyme-DNA complex to license rapid cleavage. To validate and visualize the basis for such a mechanism, we have determined the catalytic behavior of a bifunctional Type IIL restriction-modification ( RM) enzyme (DrdV) and determined its high-resolution structure at several different stages of assembly and coordination with multiple bound DNA targets using CryoEM. The structures demonstrate a mechanism of cleavage by which an initial dimer is formed between two DNA-bound enzyme molecules, positioning the single endonuclease domain from each enzyme against the others DNA and requiring further oligomerization through differing protein-protein contacts of additional DNA-bound enzyme molecules to enable cleavage. The analysis explains how endonuclease activity is licensed by the presence of multiple target-containing DNA duplexes and provides a clear view of the assembly through 3D space of a DNA-bound RM enzyme synapse that leads to rapid cleavage of foreign DNA.

biochemistry↗