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

<jats:title>ABSTRACT</jats:title> <jats:p> 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. <jats:named-content content-type="genus-species">Pseudomonas putida</jats:named-content> , 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 phylum <jats:named-content content-type="genus-species">Proteobacteria</jats:named-content> was identified as transconjugants for pBP136 by both culture-dependent and culture-independent methods. Transconjugants belonging to the phyla <jats:named-content content-type="genus-species">Actinobacteria</jats:named-content> , <jats:named-content content-type="genus-species">Bacteroidetes</jats:named-content> , and <jats:named-content content-type="genus-species">Firmicutes</jats:named-content> were detected only by the culture-independent method. Members of the genus <jats:named-content content-type="genus-species">Pseudomonas</jats:named-content> (class <jats:named-content content-type="genus-species">Gammaproteobacteria</jats:named-content> ) were identified as major transconjugants of pCAR1 and NAH7 by both methods, whereas <jats:named-content content-type="genus-species">Delftia</jats:named-content> species (class <jats:named-content content-type="genus-species">Betaproteobacteria</jats:named-content> ) were detected only by the culture-independent method. The transconjugants represented a minority of the soil bacteria. Although pCAR1-containing <jats:named-content content-type="genus-species">Delftia</jats:named-content> strains could not be cultivated after a one-to-one filter mating assay between the donor and cultivable <jats:named-content content-type="genus-species">Delftia</jats:named-content> strains as recipients, fluorescence <jats:italic>in situ</jats:italic> hybridization detected pCAR1-containing <jats:named-content content-type="genus-species">Delftia</jats:named-content> cells, suggesting that <jats:named-content content-type="genus-species">Delftia</jats:named-content> was a “transient” host of pCAR1. </jats:p>