Single-Cell Analyses Revealed Transfer Ranges of IncP-1, IncP-7, and IncP-9 Plasmids in a Soil Bacterial Community

The conjugative transfer ranges of three different plasmids of the incompatibility groups IncP-1 (pBP136), IncP-7 (pCAR1), and IncP-9 (NAH7) were investigated in soil bacterial communities by culture-dependent and culture-independent methods.Pseudomonas putida , a donor of each plasmid, was mated with soil bacteria, and green fluorescent protein (GFP), encoded on the plasmid, was used as a reporter protein for successful transfer. GFP-expressing transconjugants were detected and separated at the single-cell level by flow cytometry. Each cell was then analyzed by PCR and sequencing of its 16S rRNA gene following either whole-genome amplification or cultivation. A large number of bacteria within the phylumProteobacteria was identified as transconjugants for pBP136 by both culture-dependent and culture-independent methods. Transconjugants belonging to the phylaActinobacteria ,Bacteroidetes , andFirmicutes were detected only by the culture-independent method. Members of the genusPseudomonas (classGammaproteobacteria ) were identified as major transconjugants of pCAR1 and NAH7 by both methods, whereasDelftia species (classBetaproteobacteria ) were detected only by the culture-independent method. The transconjugants represented a minority of the soil bacteria. Although pCAR1-containingDelftia strains could not be cultivated after a one-to-one filter mating assay between the donor and cultivableDelftia strains as recipients, fluorescencein situ hybridization detected pCAR1-containingDelftia cells, suggesting thatDelftia was a “transient” host of pCAR1.