Biomonitoring of pJP4-carrying Pseudomonas chlororaphis with Trb protein-specific antisera.

ABSTRACT

The transfer of catabolic genes on conjugative plasmids to indigenous organisms from which they may spread further into the community allows the introduction of new biodegradative pathways for metabolic conversion of pollutants to the community. Biomonitoring of IncP plasmid pJP4-carrying Pseudomonas chlororaphis from the rhizosphere of Arabidopsis thaliana was achieved using antisera specific for proteins from the plasmid transfer machinery. Antisera were generated that recognized TrbC and TrbF, the putative major and minor components of pJP4-determined pili, respectively, and the putative lipoprotein TrbH. Cell fractionation studies showed association of TrbC, TrbF and TrbH with the cells and suggested that TrbC and TrbF are part of extracellular pJP4-determined pili. TrbF and TrbH antisera allowed specific detection of IncP compared with IncN or IncW plasmid-carrying cells and even permitted differentiation between bacteria carrying IncPalpha plasmid RP4 and IncPbeta plasmid pJP4. Immunofluorescence microscopy was applied to detect TrbF and TrbH signal at the cell periphery, allowing distinction from autofluorescing cells and soil debris. In situ experiments showed specific recognition of pJP4-carrying cells from laboratory cultures, as well as from the rhizosphere of A. thaliana grown in natural soil. After co-inoculation of donor P. chlororaphis pJP4 and recipient Ralstonia eutropha, a combination of immunofluorescence and oligonucleotide hybridization techniques permitted the detection of plasmid transfer between both organisms in the A. thaliana rhizosphere. This strategy may be generally applicable for the analysis of plasmid transfer in natural ecosystems.