Biology subjects

Scanlan, D.

Publications and source records attributed to Scanlan, D..

8 recordsLinked to original sources

bioRxiv2025

Rolling out plaque-2-sequence: a single plaque sequencing approach enabling rapid, low-cost sequencing of phages directly from plaques

Rapid, accurate, and scalable sequencing of bacteriophage genomes is critical to advance phage therapy, build phage biobanks and understand phage genomic diversity. Current methods are based on sequencing and assembling complete bacteriophage genomes using short- or long-read technologies. However, current protocols require large DNA input and are cost prohibitive which limits their application to phage collections that typically are large and have low-biomass. In order to address this we have developed plaque-2-sequence, a robust and cost-effective workflow for high-throughput phage genome sequencing that will transform the speed and cost of attaining phage genomes. Plaque-2-sequence combines low-input transposase-based library preparation, amplification, nanopore sequencing and optimised assembly steps tailored to phage genomes. We applied the method to phages isolated on seven genetically diverse bacterial hosts; Escherichia, Pseudomonas, Synechococcus, Enterococcus, Klebsiella , Serratia and Enterobacter. High quality genome assemblies were validated using CheckV and benchmarking against previously sequenced phage isolates. Compared to standard Illumina sequencing, plaque-2-sequence offers [~]10-fold savings in sequencing price for individual labs. Furthermore, it substantially decreases the time required to produce a phage genome, once a plaque is obtained. Offering the ability to routinely obtain hundreds of phage genome sequences a week, with minimal hands-on time. Plaque-2-sequence enables systematic genomic characterisation of phage isolates, facilitating taxonomic classification, for the development of large scale phage biobanks. Impact StatementHere we have optimised a method for high-throughput sequencing of bacteriophage genomes from single plaques (plaque-2-sequence). We present a robust, high-throughput and cost-effective workflow. Plaque-2-sequence combines low-input transposase-based library preparation, amplification, nanopore sequencing and optimised assembly steps tailored to phage genomes. We demonstrate the scalability of this approach by sequencing over 100 phages from multiple bacterial hosts. This marks a step-change for the field, allowing phage genome sequencing to keep pace with phage isolation rates, and transforming how rapidly we can explore and understand phage genomic diversity.

microbiology↗

bioRxiv2023

Glyceroglycolipids are essential for Burkholderia cenocepacia intracellular survival by preventing phagolysosome acidification

Burkholderia cenocepacia is a problematic pathogen that infects people with cystic fibrosis and often causes fatal "cepacia syndrome". B. cenocepacia infection is difficult to treat due to the high intrinsic resistance of the bacterium to antimicrobials and its ability to survive in macrophages. In this study, we uncover a hitherto unknown aspect of B. cenocepacias pathogenesis related to the formation of new glyceroglycolipids, which is required for intracellular survival. Using lipidomics, we observed that B. cenocepacia can produce three glyceroglycolipid species in phosphate deplete conditions using a PlcP-mediated lipid remodelling pathway originally discovered in soil and ocean-dwelling bacteria. While lipid remodelling as an adaptation strategy for environmental microbes to cope with the scarcity of phosphorus is known, its role in intracellular bacterial survival was not investigated. Using mammalian macrophages and Galleria mellonella larvae as infection models, we showed that the mutant unable to perform membrane lipid remodelling ({Delta}plcP) could not establish infection. Unlike the wild type bacterium, the {Delta}plcP mutant did not replicate within macrophages and failed to prevent phagosome acidification. Comparative genomics analyses showed that this PlcP pathway is conserved in all pathogenic Burkholderia that infect a variety of mammalian and plant hosts. Overall, our results indicate that membrane lipid remodelling plays an essential, yet previously overlooked, role in subverting host immunity.

microbiology↗

bioRxiv2023

The long and short of it: Benchmarking viromics using Illumina, Nanopore and PacBio sequencing technologies

Viral metagenomics has fuelled a rapid change in our understanding of global viral diversity and ecology. Long-read sequencing and hybrid approaches that combine long and short read technologies are now being widely implemented in bacterial genomics and metagenomics. However, the use of long-read sequencing to investigate viral communities is still in its infancy. While Nanopore and PacBio technologies have been applied to viral metagenomics, it is not known to what extent different technologies will impact the reconstruction of the viral community. Thus, we constructed a mock phage community of previously sequenced phage genomes and sequenced using Illumina, Nanopore, and PacBio sequencing technologies and tested a number of different assembly approaches. When using a single sequencing technology, Illumina assemblies were the best at recovering phage genomes. Nanopore- and PacBio-only assemblies performed poorly in comparison to Illumina in both genome recovery and error rates, which both varied with the assembler used. The best Nanopore assembly had errors that manifested as SNPs and INDELs at frequencies ~4x and 120x higher than found in Illumina only assemblies respectively. While the best PacBio assemblies had SNPs at frequencies ~3.5 x and 12x higher than found in Illumina only assemblies respectively. Despite high read coverage, long-read only assemblies failed to recover a complete genome for any of the 15 phage, down sampling of reads did increase the proportion of a genome that could be assembled into a single contig. Overall the best approach was assembly by a combination of Illumina and Nanopore reads, which reduced error rates to levels comparable with short read only assemblies. When using a single technology, Illumina only was the best approach. The differences in genome recovery and error rates between technology and assembler had downstream impacts on gene prediction, viral prediction, and subsequent estimates of diversity within a sample. These findings will provide a starting point for others in the choice of reads and assembly algorithms for the analysis of viromes. Data SummaryAll reads from virome sequencing were submitted to the ENA under study PRJEB56639. The assemblies are provided via FigShare (https://figshare.com/s/2d9b5121eb421d370455). Author NotesEight Supplementary Tables and nine Supplementary Figures are available with the online version of this article.

microbiology↗

bioRxiv2021

Meta-exoproteomics identifies active plant-microbe interactions operating in the rhizosphere

The advance of DNA sequencing technologies has drastically changed our perception of the complexity and structure of the plant microbiome and its role in augmenting plant health. By comparison, our ability to accurately identify the metabolically active fraction of soil microbiota and their specific functional role is relatively limited. Here, we combined our recently developed protein extraction method and an iterative bioinformatics pipeline to enable the capture and identification of extracellular proteins (meta-exoproteomics) expressed in the rhizosphere of Brassica spp. First, we validated our method in the laboratory by successfully identifying proteins related to the host plant (Brassica rapa) and a bacterial inoculant, Pseudomonas putida BIRD-1, revealing the latter expressed numerous rhizosphere specific proteins linked to the acquisition of plant-derived nutrients. Next, we analysed natural field-soil microbial communities associated with Brassica napus L (Oil Seed rape). By combining deep-sequencing metagenomics with meta-exoproteomics, a total of 1882 proteins were identified in bulk and rhizosphere samples. Importantly, meta-exoproteomics identified a clear shift (p<0.001) in the metabolically active fraction of the soil microbiota responding to the presence of B. napus roots that was not apparent in the composition of the total microbial community (metagenome). This metabolic shift was associated with the stimulation of rhizosphere-specialised bacteria, such as Gammaproteobacteria, Betaproteobacteria and Flavobacteriia and the upregulation of plant beneficial functions related to phosphorus and nitrogen mineralisation. By providing the first meta-proteomic level assessment of the active plant microbiome at the field-scale, this study demonstrates the importance of moving past a genomic assessment of the plant microbiome in order to determine ecologically important plant: microbe interactions driving plant growth.

microbiology↗

bioRxiv2021

A highly active phosphate-insensitive phosphatase is widely distributed in nature

The regeneration of bioavailable phosphate from immobilised organophosphorus represents a key process in the global phosphorus cycle and is facilitated by enzymes known as phosphatases. Most bacteria possess at least one of three major phosphatases, known as PhoA, PhoX and PhoD, whose activity is optimal under alkaline conditions. The production and activity of these three phosphatase families is negatively regulated by phosphate availability and thus these enzymes play a major role in scavenging phosphorus only during times of phosphate scarcity. Here, we reveal a previously overlooked phosphate-insensitive phosphatase, PafA, prevalent in Bacteroidetes, which is highly abundant in nature and represents a major route for the remineralisation of phosphate in the environment. Using Flavobacterium johnsoniae as the model, we reveal PafA is highly active towards phosphomonoesters. Unlike other major phosphatases, PafA is fully functional in the presence of its metabolic product, phosphate, and is essential for growth on phosphorylated carbohydrates as a sole carbon source. PafA, which is constitutively produced under all growth conditions tested, rapidly remineralises phosphomonoesters producing significant quantities of bioavailable phosphate that can cross feed into neighbouring cells. pafA is both abundant and highly expressed in the global ocean and abundant in plant rhizospheres, highlighting a new and important enzyme in the global phosphorus cycle with applied implications for agriculture as well as biogeochemical cycling. We speculate PafA expands the metabolic niche of Bacteroidetes by enabling utilisation of abundant organophosphorus substrates in the presence of excess phosphate, when other microbes are rendered incapable. Significance statementPhosphorus is an essential element for all life on Earth. Global primary production, and thus the ability for oceans and soils to drawdown atmospheric carbon dioxide, is in part controlled by the availability of inorganic phosphate. Likewise, global food production is also reliant on adequate supplies of phosphorus to both plants and animals. A major fraction of the total phosphorus pool exists as organic phosphorus, which requires mineralisation to phosphate prior to incorporation into cellular biomolecules. This important process is performed by enzymes known as phosphatases. Here, we reveal that the unique bacterial phosphatase, PafA, is a key player in the global phosphorus cycle and presents a major route for the regeneration of bioavailable phosphate required for both primary and secondary production.

microbiology↗

bioRxiv2020

Lipidomic analysis of roseobacters of the pelagic RCA cluster and their response to phosphorus limitation

The marine roseobacter-clade Affiliated cluster (RCA) represents one of the most abundant groups of bacterioplankton in the global oceans, particularly in temperate and sub-polar regions. They play a key role in the biogeochemical cycling of various elements and are important players in oceanic climate-active trace gas metabolism. In contrast to copiotrophic roseobacter counterparts such as Ruegeria pomeroyi DSS-3 and Phaeobacter sp. MED193, RCA bacteria are truly pelagic and have smaller genomes. We have previously shown that RCA bacteria do not appear to encode the PlcP-mediated lipid remodelling pathway, whereby marine heterotrophic bacteria remodel their membrane lipid composition in response to phosphorus (P) stress by substituting membrane glycerophospholipids with alternative glycolipids or betaine lipids. In this study, we report lipidomic analysis of six RCA isolates. In addition to the commonly found glycerophospholipids such as phosphatidylglycerol and phosphatidylethanolamine, RCA bacteria synthesise a relatively uncommon phospholipid, acylphosphatidylglycerol, which is not found in copiotrophic roseobacters. Instead, like the abundant SAR11 clade, RCA bacteria upregulate ornithine lipid biosynthesis in response to P stress, suggesting a key role of this aminolipid in the adaptation of marine heterotrophs to oceanic nutrient limitation.

microbiology↗

bioRxiv2019

Novel mechanisms for phosphate acquisition in abundant rhizosphere-dwelling Bacteroidetes

Global food production is reliant on the application of finite phosphorus (P) fertilisers. Numerous negative consequences associated with intensive P fertilisation have resulted in a high demand to find alternative sustainable methods that will enhance crop P uptake. Bacteroidetes, primarily from the genus Flavobacterium, have recently been shown to be abundant members of the plant microbiome, but their general ecological role and potential to mobilise P in the rhizosphere remains very poorly characterised. Here, we sought to determine the P mobilisation potential of Flavobacterium strains isolated from the rhizosphere of oilseed rape (Brassica napus L.). We show that these Flavobacterium strains possess novel mechanisms for P mobilisation and subsequent acquisition. These include the constitutive and inducible expression of completely novel and phylogenetically distinct phosphatases, the phosphate starvation inducible expression of uncharacterised and hypothetical genes and gene clusters and, for the first time, the involvement of outer membrane SusCD transport complexes (usually associated with carbohydrate transport) in P acquisition. The genes encoding these unusual phosphate starvation inducible proteins were enriched in plant-associated Flavobacterium strains suggesting that this machinery represents niche-adaptive strategies for overcoming P scarcity in this genus. We propose that abundant rhizosphere-dwelling Flavobacterium spp. have evolved unique mechanisms for coping with Pi-stress which may provide novel solutions for future sustainable agricultural practices.

microbiology↗

bioRxiv2018

Riding the wave of genomics, to investigate aquatic coliphage diversity and activity

Bacteriophages infecting Escherichia coli have been used as a proxy for faecal matter and water quality from a variety of environments. However, the diversity of coliphages that are present in seawater remains largely unknown, with previous studies largely focusing on morphological diversity. Here, we isolated and characterised coliphages from three coastal locations in the UK and Poland. This revealed a surprising genetic diversity, with comparative genomics and phylogenetic analysis of phage isolates facilitating the identification of putative new species within the genera RB69virus and T5virus and a putative new genus within the subfamily Tunavirinae. Furthermore, by combining this genomic data with proteomic and host range analyses a number of phage structural proteins were identified, one of which is likely to be responsible for the observed differences in host range.

microbiology↗