Dosage of Tn916 circular intermediates in Enterococcus faecalis

Upon excision, conjugative transposon Tn916 forms a nonreplicating circular intermediate (CI), which is essential both for transposition and conjugal transfer. In this work we developed an assay to quantify the circular forms of Tn916. By this method we defined a new measurable trait of Tn916-carrying strains: the "CI copy number." CI dosage was performed by nested PCR (with primers designed on Tn916 termini) in a limiting dilution assay, where Poisson statistics were used to calculate the number of PCR amplification targets from the proportion of the negative endpoints. The number of CI was normalized to the number of bacterial chromosomes. This method enabled us to study the relationship between CI copy number, presence of tetracycline (the resistance marker of Tn916) in the culture medium, and conjugation frequency. Three isogenic strains of Enterococcus faecalis OG1 with Tn916 inserted at different sites on the chromosome were investigated for CI content. CI copy number varied depending on the strain, ranging between 7.8 and 610 copies per 10(6) chromosomes. Growth in liquid media containing tetracycline provoked an important increase both in CI copy number and conjugation frequency. This effect was more marked in low-frequency donors. While cell-cell contact during filter mating did not produce an increase in CI copy number, Tn916 conjugation frequency was found to be dependent on CI copy number in donor cells. The dose-response curve showed a linear relationship with a slope of 0.74, for the entire range of conjugation frequencies tested (from 5.1 x 10(-8) to 2.8 x 10(-6) transconjugants per donor.