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

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 the use of non-genetically tractable strains. Here, we applied auxotrophic donor counter-selection in a relatively high-throughput and accessible screening format to characterize PHR across a diverse collection of environmental isolates without the need for recipient engineering. Specifically, we used two auxotrophic donors (Pseudomonas fluorescens and Pseudomonas putida) and plasmid pQBR57-tphKAB, an environmental plasmid engineered for terephthalic acid bioremediation. We screened a library of 101 soil isolates as potential recipients, including genera such as Pseudomonas, Bacillus and Xanthomonas. We only observed conjugation into other Pseudomonas, but donor identity was found to affect PHR, with P. fluorescens conjugating the plasmid into more recipient strains than P. putida. Phylogenomic analysis revealed that transconjugants clustered primarily with the Pseudomonas citronellolis lineage, previously isolated from soil. In strains that were close relatives of transconjugants but unable to acquire the plasmid, we observed five defence systems not present in transconjugants that may act as barriers to plasmid acquisition. Our approach demonstrates how auxotrophic donor counter-selection can be deployed at scale to screen PHR in environmental isolates and to investigate the influence of donor identity on plasmid conjugation.