Using auxotrophic donor strains to explore pQBR57 plasmid host range among environmental soil bacterial isolates

Alejandro Marquiegui-Alvaro, Anastasia Kottara, Matthew J. N. Thomas, Alberto Scarampi, Micaela Chacón, Michael Brockhurst, Neil Dixon
2026 · doi:10.64898/2026.02.11.702040
preprint OA: closed CC-BY-ND-4.0
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Abstract

Plasmid host range (PHR) plays a key role in the spread of ecologically important genes, alongside applications in microbiome engineering, and environmental biotechnology. PHR is a complex trait arising from the combination of plasmid, donor and recipient properties. Most studies of PHR use a single donor strain, leaving the role of the donor unexplored, and often require genetically tagged recipient strains for counter selection, which limits use of non-genetically tractable strains. Here we developed a PHR screening method using auxotrophic donors that bypasses the need to genetically tag recipients, thus allowing the screening of culturable environmental bacterial strains. Specifically, we used two auxotrophic donors ( P. fluorescens and P. putida ), and the plasmid pQBR57-tphKAB, an environmental plasmid engineered for terephthalic acid bioremediation. We screened a library of 101 soil isolates, as potential recipients, including common soil genera of soil bacteria, Pseudomonas, Bacillus and Xanthomonas . We only observed conjugation into other Pseudomonas , but donor identity affected PHR, with P. fluorescens conjugating the plasmid into more recipient strains than P. putida . Phylogenomic analysis revealed that transconjugants clustered with P. citronellosis and P. putida lineages. In strains that were close relatives of transconjugants but who were unable to acquire the plasmid, we observed 5 defence systems not present in transconjugants that may act as barriers to plasmid acquisition. Our method provides a rapid, tag-free framework for screening PHR in environmental isolates and for investigating the influence of donor identity on plasmid conjugation.
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Abstract Plasmid host range (PHR) plays a key role in the spread of ecologically important genes, alongside applications in microbiome engineering, and environmental biotechnology. PHR is a complex trait arising from the combination of plasmid, donor and recipient properties. Most studies of PHR use a single donor strain, leaving the role of the donor unexplored, and often require genetically tagged recipient strains for counter selection, which limits use of non-genetically tractable strains. Here we developed a PHR screening method using auxotrophic donors that bypasses the need to genetically tag recipients, thus allowing the screening of culturable environmental bacterial strains. Specifically, we used two auxotrophic donors (P. fluorescens and P. putida), and the plasmid pQBR57-tphKAB, an environmental plasmid engineered for terephthalic acid bioremediation. We screened a library of 101 soil isolates, as potential recipients, including common soil genera of soil bacteria, Pseudomonas, Bacillus and Xanthomonas. We only observed conjugation into other Pseudomonas, but donor identity affected PHR, with P. fluorescens conjugating the plasmid into more recipient strains than P. putida. Phylogenomic analysis revealed that transconjugants clustered with P. citronellosis and P. putida lineages. In strains that were close relatives of transconjugants but who were unable to acquire the plasmid, we observed 5 defence systems not present in transconjugants that may act as barriers to plasmid acquisition. Our method provides a rapid, tag-free framework for screening PHR in environmental isolates and for investigating the influence of donor identity on plasmid conjugation. Competing Interest Statement The authors have declared no competing interest.

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License: CC-BY-ND-4.0