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Dosage of Tn916 circular intermediates in Enterococcus faecalis.
Plasmid 1995; 34(1):48-57P

Abstract

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.

Authors+Show Affiliations

Dipartimento di Biologia Molecolare, Università di Siena, Italy.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

7480170

Citation

Manganelli, R, et al. "Dosage of Tn916 Circular Intermediates in Enterococcus Faecalis." Plasmid, vol. 34, no. 1, 1995, pp. 48-57.
Manganelli R, Romano L, Ricci S, et al. Dosage of Tn916 circular intermediates in Enterococcus faecalis. Plasmid. 1995;34(1):48-57.
Manganelli, R., Romano, L., Ricci, S., Zazzi, M., & Pozzi, G. (1995). Dosage of Tn916 circular intermediates in Enterococcus faecalis. Plasmid, 34(1), pp. 48-57.
Manganelli R, et al. Dosage of Tn916 Circular Intermediates in Enterococcus Faecalis. Plasmid. 1995;34(1):48-57. PubMed PMID: 7480170.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Dosage of Tn916 circular intermediates in Enterococcus faecalis. AU - Manganelli,R, AU - Romano,L, AU - Ricci,S, AU - Zazzi,M, AU - Pozzi,G, PY - 1995/7/1/pubmed PY - 1995/7/1/medline PY - 1995/7/1/entrez SP - 48 EP - 57 JF - Plasmid JO - Plasmid VL - 34 IS - 1 N2 - 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. SN - 0147-619X UR - https://www.unboundmedicine.com/medline/citation/7480170/Dosage_of_Tn916_circular_intermediates_in_Enterococcus_faecalis_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0147-619X(85)71032-3 DB - PRIME DP - Unbound Medicine ER -