Conjugative transfer of Tn916 in Enterococcus faecalis: trans activation of homologous transposons

Abstract

tet. Experiments showed that the presence of one element (e.g., Tn916AE) in a recipient cell did not prevent the uptake of the other (e.g., Tn916) (7, 24), indicating an absence of negatively acting factors relating to entry exclusion or incompatibility. The presence of a transposon on the recipient chromosome also failed to prevent the zygotically induced excision-insertion that occurs at a high frequency when Tn916 enters passively on a conjugative plasmid (7). The data argue against the presence of a strong, trans-acting negative regulator being produced by an established element.In the present study we focus on the interaction that occurs between Tn916 and Tn9J6AE when they reside together in a donor cell. Evidence is presented showing that the transfer of one element results in a high-frequency trans activation of the other. Comobilization of resident plasmids was also detected, but at frequencies much lower than that involving the transposon. MATERIALS AND METHODSBacterial strains, plasmids, and media. The bacterial strains and plasmids used in this study are listed in Tables 1 and 2, respectively. Cultures of E. faecalis were grown in Todd-Hewitt broth (THB; Difco) at 37°C. Escherichia coli cultures were grown at 37°C in LB medium (11) for preparation of competent cells and in THB for plasmid isolation. Antibiotics, when present, were used in the following concentrations except as noted: tetracycline, 10 ,ig/ml (to maintain pAM420 in E. coli, a concentration of 60 p.g/ml was used); erythromycin, 10 ,ug/ml; chloramphenicol, 25 ,ug/ml; fusidic acid, 25 p.g/ml; streptomycin, 1,000 jig/ml; spectinomycin, 500 ,ug/ml; rifampin, 25 ,ug/ml. Rifampin was from Calbiochem, whereas all other antibiotics were from Sigma.DNA analyses. Chromosomal DNA was prepared by a standard protocol (35). Plasmid DNA was purified by an alkaline lysis procedure modified from that of Ish-Horowicz 7136 JOURNAL

Use of an excision reporter plasmid to study the intracellular mobility of the conjugative transposon Tn916 in Gram-positive bacteria

“…The host range of transfer of Tn916 might depend on the donor potential of the transposon-delivery strain : only strains having a high donor potential can undergo conjugative transposition from Gram-positive to Gram-negative bacteria (Poyart et al, 1995). Tn916 insertions located at different sites of the bacterial genome may possess different donor potentials when considered independently and it has been suggested that the insertion with the greatest potential would be the most likely to excise initially and that this event could stimulate, by trans-activation, movements of all other transposons present in the cell (Flannagan & Clewell, 1991). The dosage of Tn916 circular intermediates in E. faecalis performed by quantitative PCR assay has revealed that the frequency of Tn916 conjugation is dependent on the copy number of circular intermediates present in the donor cell population (Manganelli et al,199.5).…”

Amplification of the Tetracycline Resistance Determinant of pAMα1 in Enterococcus faecalis Requires a Site-Specific Recombination Event Involving Relaxase

“…pAM␣1 was cleaved with SalI, and the larger of the two SalI fragments (Fig. 1) was religated and introduced into JH2-2 by electroporation (16). pAM8502 is a mutant with a 789-bp deletion in mobB.…”

A functional origin of transfer (oriT) on the conjugative transposon Tn916

“…The approach to identifying an oriT on Tn916 was to make use of a trans-activation system based on the previously described phenomenon (14) whereby the conjugative transfer of a given transposon activates the transfer of a homologous element located at a different site in the same cell. It was previously shown that when Tn916 was present in E. faecalis on a plasmid and a homologous element such as Tn916⌬E (which possesses an erm determinant in place of tet [27]) was present on the chromosome, selection for transconjugants obtaining one of the elements resulted in unselected acquisition of the other about 50% of the time (14). It was reasoned that if an oriT site of Tn916 could be cloned on a nonmobilizable plasmid and introduced into a bacterial host harboring an intact transposon (Tn916⌬E) on the chromosome, selection for conjugative transfer of the chromosomeborne element should result in mobilization of the recombinant plasmid.…”

“…Ligation mixes were introduced into E. coli DH5␣ by electroporation. Qiagen-prepared DNA was then used to transform electrocompetent E. faecalis cells essentially as described previously (14).…”