Sampson, H. | bioRxiv Science

bioRxiv2026

Microaerobic Copper Stress Redirects Pyruvate Metabolism and Reveals a CopL-linked Nitrogen Response in Staphylococcus aureus.

Copper is both an essential enzyme cofactor and an antimicrobial agent deployed by the host immune system to eradicate micro-organisms. The epidemic community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) lineage USA300 carries mobile genetic elements that encode copX/B and copL, conferring hyper-resistance to copper, but the role of CopL beyond extracellular copper sequestration remains unclear. We have combined RNA sequencing with targeted metabolite assays under microaerobic conditions, more reflective of host environments, to define key copper induced responses in WT and copL mutant strains. Subinhibitory copper exposure in microaerobic conditions triggered a distinctive transcriptional response across multiple biological functions. Unlike previous studies, copper exposure did not induce an oxidative stress response. Instead, classical copper resistance, teichoic acid modification, immune-evasion factors and core metabolic genes were induced, while genes for stress responses, metal homeostasis and virulence were repressed. Gene set enrichment analysis (GSEA) identified regulation by multiple global regulators, e.g. SigB, CodY, CcpA, Agr and Sae. Copper exposure affected metabolism, redirecting pyruvate flux toward acetoin and lactate production rather than acetate, accompanied by coordinated shifts in TCA cycle and amino acid pathways, including glutamate accumulation. Inactivation of copL revealed a distinct adaptive response, with strong induction of nitrogen metabolism genes and nitrite reduction. Together, these data show that copper functions as a regulatory signal, triggering coordinated transcriptional and metabolic remodelling that potentiates S. aureus fitness in the host.

microbiology↗

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↗