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Dosage of Tn916 circular intermediates in Enterococcus faecalis
R Manganelli1, L Romano, S Ricci
1Dipartimento di Biologia Molecolare, Università di Siena, Italy.
Plasmid
|July 1, 1995
Summary
Researchers developed a new assay to quantify circular forms of the Tn916 conjugative transposon (CI). CI copy number and conjugation frequency increase with tetracycline, impacting bacterial gene transfer.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Conjugative transposon Tn916 forms a circular intermediate (CI) crucial for transposition and transfer.
- Quantifying CI copy number is essential for understanding Tn916 dynamics.
Purpose of the Study:
- Develop a novel assay to quantify Tn916 circular intermediates (CI).
- Define and measure CI copy number as a new trait in bacterial strains.
- Investigate the relationship between CI copy number, tetracycline presence, and conjugation frequency.
Main Methods:
- Developed a quantitative assay using nested PCR and limiting dilution.
- Calculated CI copy number using Poisson statistics, normalized to chromosomal DNA.
- Studied three isogenic Enterococcus faecalis OG1 strains with Tn916 insertions.
Main Results:
- CI copy number varied significantly between strains (7.8-610 copies/10^6 chromosomes).
- Tetracycline in media increased both CI copy number and conjugation frequency, especially in low-frequency donors.
- Tn916 conjugation frequency showed a linear dependence on CI copy number (slope 0.74).
Conclusions:
- The developed assay effectively quantifies Tn916 CI copy number.
- Tetracycline positively regulates CI copy number and conjugation efficiency.
- CI copy number is a key determinant of Tn916 conjugal transfer efficiency.