Conjugal TOL Transfer from Pseudomonas putida to Pseudomonas aeruginosa : Effects of Restriction Proficiency, Toxicant Exposure, Cell Density Ratios, and Conjugation Detection Method on Observed Transfer Efficiencies

Abstract

The effects of restriction proficiency and premating exposure to toxicants on conjugal transfer of the TOL plasmid between Pseudomonas spp. was investigated by examinations of filter matings. A Pseudomonas putida KT2442-derived strain carrying a gfp -tagged variant of the TOL plasmid was used as a donor, and both restriction-deficient (PAO1162N) and -proficient (PAO2002N) Pseudomonas aeruginosa strains were used as recipients. The in situ enumeration of conjugation events allowed us to obtain frequency estimates that were unbiased by transconjugant growth or plasmid retransfer. We observed a strong dependence of the plasmid transfer frequency on the initial donor-to-recipient ratio of surface matings, which invalidated the use of mass action-based plasmid transfer kinetic estimators. Careful control of the initial parental cell densities permitted evaluations of the true effects of restriction proficiency and toxicant exposure on TOL transfer. At standard donor-to-recipient ratios (10 ⁻³ for PAO1162N and 2 × 10 ¹ for PAO2002N) and total cell densities (10 ⁵ cells/mm ² for PAO1162N and 10 ⁶ cells/mm ² for PAO2002N), plasmid transfer frequencies without toxicant exposure were approximately 10 ⁻⁷ (events/mm ² ) ⁻¹ for PAO1162N and 10 ⁻¹¹ (events/mm ² ) ⁻¹ for PAO2002N based on in situ observations of conjugation events. The enumeration of transconjugants via selective plating yielded transfer frequencies that were up to 1 order of magnitude lower. Premating exposure to sodium dodecyl sulfate (1 to 10 mM) significantly increased the transfer frequency for the restriction-proficient strain PAO2002N ( P < 0.05) but not for the restriction-deficient strain PAO1162N. On the other hand, premating exposure to ethanol, toluene, or phenol had no positive effect on the plasmid transfer frequency. Clearly, restriction proficiency provides a strong barrier to interspecific transfer of the TOL plasmid, and this barrier was only marginally attenuated by recipient exposure to toxicants within the ranges examined.