Biology subjects

Owen, S. V.

Publications and source records attributed to Owen, S. V..

14 recordsLinked to original sources

bioRxiv2026

Genome-wide characterization of host factors involved in single-stranded RNA and DNA phage infection pathways

Single stranded RNA (ssRNA) and single stranded DNA (ssDNA) bacteriophages represent a key component of the global virome, yet the host genetic networks supporting their infection cycles remain poorly understood. Here, we present a comprehensive, genome-wide mapping of the genetic landscape regulating infection cycles for F pilus-dependent ssRNA and ssDNA phages in Escherichia coli. Genetic screens across ssRNA phages spanning all four genogroups of the Leviviricetes revealed a highly conserved network of host dependencies, with the notable exception of the F plasmid gene traD. While primary structural receptor components and dsbA mediated disulfide bond formation are universally required across all lineages to ensure F pilus integrity, traD exhibits a strict genogroup-specific requirement during entry, showing variable essentiality across different viral groups despite sharing an identical primary receptor. Our gene dosage screens revealed that an elevated copy number of the hslU protease or the RNA chaperone stpA restricts infection, identifying clear genetic barriers that can perturb the viral life cycle. Parallel assays with filamentous ssDNA phages produced host factor profiles consistent with published literature, while revealing additional variations in host dependency. These screens confirmed that ssDNA phages strictly rely on the host TolQRA complex for entry downstream of pilus engagement. The assays tracked prominent negative fitness signatures across homeostatic clusters, highlighting how the physiological burden of continuous, non-lytic virion extrusion strains the host envelope. Finally, this comparative approach traced the selectivity of our isolation host (E. coli HSF) to a horizontally acquired capsule architecture from Klebsiella. This surface shield excludes a large panel of double stranded DNA phages isolated on diverse E. coli strains, while allowing virions from ssDNA and ssRNA phages to engage the extended F pilus and bypass the barrier via native pilus retraction. Together, this work provides a systematic, class-wide map of single stranded phage-host interactions, bridging classical genetics with modern viral discovery while establishing a robust host platform to access uncultured viral diversity and a functional blueprint to design next generation diagnostics, protein antibiotics, and biocontrol tools to halt horizontal gene transfer.

microbiology↗

bioRxiv2024

Phage DisCo: targeted discovery of bacteriophages by co-culture

Phages interact with many components of bacterial physiology from the surface to the cytoplasm. Although there are methods to determine the receptors and intracellular systems a specified phage interacts with retroactively, finding a phage that interacts with a chosen piece of bacterial physiology a priori is very challenging. Variation in phage plaque morphology does not to reliably distinguish distinct phages and therefore many potentially redundant phages may need to be isolated, purified, and individually characterized to find phages of interest. Here, we present a method in which multiple bacterial strains are co-cultured on the same screening plate to add an extra dimension to plaque morphology data. In this method, Phage Discovery by Co-culture (Phage DisCo), strains are isogenic except for fluorescent tags and one perturbation expected to impact phage infection. Differential plaquing on the strains is easily detectable by fluorescent signal and implies that the perturbation made to the surviving strain in a plaque prevents phage infection. We validate the phage DisCo method by showing that characterized phages have the expected plaque morphology on Phage DisCo plates and demonstrate the power of Phage DisCo for multiple targeted discovery applications, from receptors to phage defense systems.

microbiology↗

bioRxiv2023

Salmonella enterica serovar Typhimurium ST313 sublineage 2.2 has emerged in Malawi with a characteristic gene expression signature and a fitness advantage

Invasive non-typhoidal Salmonella (iNTS) disease is a serious bloodstream infection that targets immune-compromised individuals, and causes significant mortality in sub-Saharan Africa. Salmonella enterica serovar Typhimurium ST313 causes the majority of iNTS in Malawi, and we performed an intensive comparative genomic analysis of 608 isolates obtained from fever surveillance at the Queen Elizabeth Hospital, Blantyre between 1996 and 2018. We discovered that following the upsurge of the well-characterised S. Typhimurium ST313 lineage 2 from 1999 onwards, two new multidrug-resistant sublineages designated 2.2 and 2.3, emerged in Malawi in 2006 and 2008, respectively. The majority of S. Typhimurium isolates from human bloodstream infections in Malawi now belong to sublineage 2.2 or 2.3. To identify factors that characterised the emergence of the prevalent ST313 sublineage 2.2, we performed genomic and functional analysis of two representative strains, D23580 (lineage 2) and D37712 (sublineage 2.2). Comparative genomic analysis showed that the chromosome of ST313 lineage 2 and sublineage 2.2 were broadly similar, only differing by 29 SNPs and small indels and a 3kb deletion in the Gifsy-2 prophage region that spanned the sseI pseudogene. Lineage 2 and sublineage 2.2 have unique plasmid profiles that were verified by long read sequencing. The transcriptome was initially explored in 15 infection-relevant conditions and within macrophages. Differential gene expression was subsequently investigated in depth in the four most important in vitro growth conditions. We identified up-regulation of SPI2 genes in non-inducing conditions, and down-regulation of flagellar genes in D37712, compared to D23580. Following phenotypic confirmation of transcriptional differences, we discovered that sublineage 2.2 had increased fitness compared with lineage 2 during mixed-growth in minimal media. We speculate that this competitive advantage is contributing to the continuing presence of sublineage 2.2 in Malawi.

bioinformatics↗

bioRxiv2023

Diverse and Abundant Viruses Exploit Conjugative Plasmids

Phages exert profound evolutionary pressure on bacteria by interacting with receptors on the cell surface to initiate infection. While the majority of phages use chromosomally-encoded cell surface structures as receptors, plasmid-dependent phages exploit plasmid-encoded conjugation proteins, making their host range dependent on horizontal transfer of the plasmid. Despite their unique biology and biotechnological significance, only a small number of plasmid-dependent phages have been characterized. Here we systematically search for new plasmid-dependent phages targeting IncP and IncF plasmids using a targeted discovery platform, and find that they are common and abundant in wastewater, and largely unexplored in terms of their genetic diversity. Plasmid-dependent phages are enriched in non-canonical types of phages, and all but one of the 64 phages we isolated were non-tailed, and members of the lipid-containing tectiviruses, ssDNA filamentous phages or ssRNA phages. We show that plasmid-dependent tectiviruses exhibit profound differences in their host range which is associated with variation in the phage holin protein. Despite their relatively high abundance in wastewater, plasmid-dependent tectiviruses are missed by metaviromic analyses, underscoring the continued importance of culture-based phage discovery. Finally, we identify a tailed phage dependent on the IncF plasmid, and find related structural genes in phages that use the orthogonal type 4 pilus as a receptor, highlighting the promiscuous use of these distinct contractile structures by multiple groups of phages. Taken together, these results indicate plasmid-dependent phages play an under-appreciated evolutionary role in constraining horizontal gene transfer via conjugative plasmids.

microbiology↗

bioRxiv2022

Swapped genetic code blocks viral infections and gene transfer

Removing cellular transfer RNAs (tRNAs), making their cognate codons unreadable, creates a genetic firewall that prevents viral replication and horizontal gene transfer. However, numerous viruses and mobile genetic elements encode parts of the translational apparatus, including tRNAs, potentially rendering a genetic-code-based firewall ineffective. In this paper, we show that such horizontally transferred tRNA genes can enable viral replication in Escherichia coli cells despite the genome-wide lack of three codons and the previously essential cognate tRNAs and release factor 1. By repurposing viral tRNAs, we then develop recoded cells bearing an amino-acid-swapped genetic code that reassigns two of the six serine codons to leucine during translation. This amino-acid-swapped genetic code renders cells completely resistant to viral infections by mistranslating viral proteomes and prevents the escape of synthetic genetic information by engineered reliance on serine codons to produce leucine-requiring proteins. Finally, we also repurpose the third free codon to biocontain this virus-resistant host via dependence on an amino acid not found in nature.

synthetic biology↗

bioRxiv2022

Parallel changes in gene essentiality over 50,000 generations of evolution

As evolving populations accumulate mutations, the benefits and costs of subsequent mutations change. As fitness increases, the relative benefit of new mutations typically decreases. However, the question remains whether deleterious mutations tend to have larger or smaller costs as a population adapts; theory and experiments provide support for both conflicting hypotheses. To address this question, we compared the effects of insertion mutations in every gene in Escherichia coli between ancestral and 12 independently derived strains after 50,000 generations in a uniform environment. We found both increases and decreases in the fitness costs of mutations, leaving the overall distribution of effects largely unchanged. However, at the extreme, more genes became essential over evolution than vice versa. Both changes in fitness effects and essentiality evolved in parallel across the independent populations, and most changes were not explained by structural variation or altered gene expression. Thus, the macroscopic features of the local fitness landscape remained largely unchanged, even as access to particular evolutionary trajectories changed consistently during adaptation to the experimental environment. One Sentence SummaryLimdi et al. report parallel changes in the cost of mutations in replicate lineages of a decades-long E. coli evolution experiment.

evolutionary biology↗

bioRxiv2021

The gut bacterial natural product colibactin triggers induction of latent viruses in diverse bacteria

Colibactin is a chemically unstable small molecule genotoxin produced by multiple different bacteria, including members of the human gut microbiome.1,2 While the biological activity of colibactin has been extensively investigated in mammalian systems,3 little is known about its effects on other microorganisms. Here, we discover that colibactin targets bacteria carrying prophages, inducing lytic development via the bacterial SOS response. DNA, added exogenously, protects bacteria from colibactin, as does expressing a colibactin resistance protein (ClbS) in non-colibactin-producing cells. The prophage-inducing effects we observe apply broadly across taxonomically diverse phage-bacteria systems. Finally, we identify bacteria that possess colibactin resistance genes but lack colibactin biosynthetic genes. Many of these bacteria are infected with predicted prophages, and we show that the expression of their ClbS homologs provides immunity from colibactin-triggered induction. Our study reveals a mechanism by which colibactin production could impact microbiomes and highlights an underappreciated role for microbial natural products in influencing population-level events such as phage outbreaks.

microbiology↗

bioRxiv2020

Prophage-encoded phage defence proteins with cognate self-immunity

Temperate phages are pervasive in bacterial genomes, existing as vertically-inherited islands called prophages. Prophages are vulnerable to the predation of their host bacterium by exogenous phages. Here we identify BstA, a novel family of prophage-encoded phage defense proteins found in diverse Gram-negative bacteria. BstA drives potent suppression of phage epidemics through abortive infection. During lytic replication, the bstA-encoding prophage is not itself inhibited by BstA due to a self-immunity mechanism conferred by the anti-BstA (aba) element, a short stretch of DNA within the bstA locus. Inhibition of phage replication by distinct BstA proteins from Salmonella, Klebsiella and Escherichia prophages is functionally interchangeable, but each possesses a cognate aba element. The specificity of the aba element ensures that immunity is exclusive to the replicating prophage, and cannot be exploited by heterologous BstA-encoding phages. BstA allows prophages to defend host cells against exogenous phage attack, without sacrificing their own lytic autonomy.

microbiology↗

bioRxiv2020

Forensic microbial system for high-resolution object provenance

Mapping where an object has been is a fundamental challenge for human health, commerce, and food safety. Location-specific microbes offers the potential to cheaply and sensitively determine object provenance. We created a synthetic, scalable microbial spore system that identifies object provenance in under one hour at meter-scale resolution and near single spore sensitivity, and that can be safely introduced into and recovered from the environment. This system solves the key challenges in object provenance: persistence in the environment, scalability, rapid and facile decoding, and biocontainment. Our system is compatible with SHERLOCK, facilitating its implementation in a wide range of applications.

bioengineering↗

bioRxiv2019

A window into lysogeny: Revealing temperate phage biology with transcriptomics

Integrated phage elements, known as prophages, are a pervasive feature of bacterial genomes. Prophages can enhance the fitness of their bacterial hosts by conferring beneficial functions, such as virulence, stress tolerance or phage resistance, which are encoded by accessory loci. Whilst the majority of phage-encoded genes are repressed during lysogeny, accessory loci are often highly expressed. However, novel prophage accessory loci are challenging to identify based on DNA sequence data alone. Here, we use bacterial RNA-seq data to examine the transcriptional landscapes of five Salmonella prophages. We show that transcriptomic data can be used to heuristically enrich for prophage features that are highly expressed within bacterial cells and often represent functionally-important accessory loci. Using this approach we identify a novel anti-sense RNA species in prophage BTP1, STnc6030, which mediates superinfection exclusion of phage BTP1 and immunity to closely-related phages. Bacterial transcriptomic datasets are a powerful tool to explore the molecular biology of temperate phages.

microbiology↗

bioRxiv2019

African Salmonella Typhimurium sequence type 313 lineage 2 evades MAIT cell recognition by overexpressing RibB

Mucosal-associated invariant T (MAIT) cells are a subset of innate T lymphocytes activated by bacteria that produce vitamin B2 metabolites. Mouse models of infection have demonstrated a role for MAIT cells in antimicrobial defence. However, proposed protective roles of MAIT cells in human infections remain unproven and clinical conditions associated with a selective absence of MAIT cells have not been identified. We report that typhoidal and non-typhoidal S. enterica strains generally activate MAIT cells. However, African invasive disease-associated multidrug-resistant S. Typhimurium sequence type 313 lineage 2 strains escape MAIT cell recognition through overexpression of ribB, a bacterial gene that encodes the 4-dihydroxy-2-butanone 4-phosphate synthase enzyme of the riboflavin biosynthetic pathway. This MAIT cell-specific phenotype did not extend to other innate lymphocytes. We propose that ribB overexpression is an evolved trait that facilitates evasion from immune recognition by MAIT cells and contributes to the invasive pathogenesis of S. Typhimurium sequence type 313 lineage 2 in vivo.

immunology↗

bioRxiv2019

The fitness landscape of the African Salmonella Typhimurium ST313 strain D23580 reveals unique properties of the pBT1 plasmid

We have used a transposon insertion sequencing (TIS) approach to establish the fitness landscape of the African Salmonella enterica serovar Typhimurium ST313 strain D23580, to complement our previous comparative genomic and functional transcriptomic studies. We used a genome-wide transposon library with insertions every 10 nucleotides to identify genes required for survival and growth in vitro and during infection of murine macrophages. The analysis revealed genomic regions important for fitness under two in vitro growth conditions. Overall, 724 coding genes were required for optimal growth in LB medium, and 851 coding genes were required for growth in SPI-2-inducing minimal medium. These findings were consistent with the essentiality analyses of other S. Typhimurium ST19 and S. Typhi strains. The global mutagenesis approach also identified 60 sRNAs and 413 intergenic regions required for growth in at least one in vitro growth condition. By infecting murine macrophages with the transposon library, we identified 68 genes that were required for intra-macrophage replication but did not impact fitness in vitro. None of these genes were unique to S. Typhimurium D23580, consistent with a high conservation of gene function between S. Typhimurium ST313 and ST19 and suggesting that novel virulence factors are not involved in the interaction of strain D23580 with murine macrophages. We discovered that transposon insertions rarely occurred in many pBT1 plasmid-encoded genes (36), compared with genes carried by the pSLT-BT virulence plasmid and other bacterial plasmids. The key essential protein encoded by pBT1 is a cysteinyl-tRNA synthetase, and our enzymological analysis revealed that the plasmid-encoded CysRSpBT1 had a lower ability to charge tRNA than the chromosomally-encoded CysRSchr enzyme. The presence of aminoacyl-tRNA synthetases in plasmids from a range of gram-negative and gram-positive bacteria suggests that plasmid-encoded essential genes are more common than had been appreciated.

microbiology↗

bioRxiv2018

Adding function to the genome of African Salmonella ST313

Salmonella Typhimurium ST313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580, and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe, revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in sixteen infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium SalComD23580: bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by 3 independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolise this carbon source has been negatively selected during ST313 evolution. The data revealed a novel plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multi-condition analyses to pinpoint key phenotypic differences between bacterial pathovariants.

microbiology↗

bioRxiv2017

The role of a single non-coding nucleotide in the evolution of an epidemic African clade of Salmonella

Introductory ParagraphSalmonella enterica serovar Typhimurium ST313 is a relatively newly emerged sequence type that is causing a devastating epidemic of bloodstream infections across sub-Saharan Africa. Analysis of hundreds of Salmonella genomes has revealed that ST313 is closely-related to the ST19 group of S. Typhimurium that cause gastroenteritis across the world. The core genomes of ST313 and ST19 vary by just 1000 single-nucleotide polymorphisms (SNPs). We hypothesised that the phenotypic differences that distinguish African Salmonella from ST19 are caused by certain SNPs that directly modulate the transcription of virulence genes.\n\nHere we identified 3,597 transcriptional start sites (TSS) of the ST313 strain D23580, and searched for a gene expression signature linked to pathogenesis of Salmonella. We identified a SNP in the promoter of the pgtE gene that caused high expression of the PgtE virulence factor in African S. Typhimurium, increased the degradation of the factor B component of human complement, contributed to serum resistance and modulated virulence in the chicken infection model. The PgtE protease is known to mediate systemic infection in animal models. We propose that high levels of expression PgtE of by African S. Typhimurium ST313 promotes bacterial survival and bacterial dissemination during human infection.\n\nOur finding of a functional role for an extra-genic SNP shows that approaches used to deduce the evolution of virulence in bacterial pathogens should include a focus on non-coding regions of the genome.

molecular biology↗