Tn5381, a conjugative transposon identifiable as a circular form in Enterococcus faecalis

Abstract

We have identified two 19-kb conjugative transposons (Tn5381 and Tn5383) in separate strains of multiply resistant Enterococcusfaecalis. These transposons confer resistance to tetracycline and minocycline via a tetM gene, are capable of both chromosomal and plasmid integration in a Rec-environment, and transfer between strains in the absence of detectable plasmid DNA at frequencies ranging from < 1 x 10-9 to 2 x 10'S per donor CFU, depending on the donor strain and the growth conditions. Hybridization studies indicate that these transposons are closely related to Tn916. We have identified bands of ca. 19 kb on agarose gel separations of alkaline lysis preparations from E. faecalis strains containing chromosomal copies of Tn5381, which we have confirmed to be a circularized form of this transposon. This phenomenon has previously been observed only when Tn916 has been cloned in Escherichia coli. Overnight growth of donor strains in the presence of subinhibitory concentrations of tetracycline results in an approximately 10-fold increase in transfer frequency of Tn5381 into enterococcal recipients and an increase in the amount of the circular form of Tn5381 as detectable by hybridization. These results suggest that Tn5381 is a Tn916-related conjugative transposon for which the appearance of a circular form and the conjugative-transfer frequency are regulated by a mechanism(s) affected by the presence of tetracycline in the growth medium.Conjugative transposons have been implicated in the dissemination of antimicrobial resistance among numerous gram-positive genera (4, 6). Tn916, a 16.4-kb conjugative transposon encoding resistance to tetracycline and minocycline, was first described for a strain of Enterococcusfaecalis in 1980 (12). Since that time, numerous similar elements have been described elsewhere (1,4,9,14). Common to all of these elements are a preference for integration into the bacterial chromosome, the ability to transfer between strains in the absence of detectable plasmid DNA, and the ability to alter the hemolytic properties of hemolysin-and bacteriocinproducing plasmids. With few exceptions (1, 21), conjugative transposons have encoded a tetM tetracycline and minocycline resistance determinant. In most cases, these elements have been shown to have a broad host range within gram-positive genera.On the basis of the apparent precise excision and insertion without target duplication of Tn916, a novel mechanism of transposition involving a nonreplicative circular intermediate was proposed elsewhere (13). Evidence to support the hypothesis of this mechanism includes the identification of a supercoiled form of Tn916 within an Escherichia coli strain which had been transformed with a Tn916-containing chimeric cosmid. This supercoiled molecule was able to transform Bacillus subtilis and subsequently transfer tetracycline resistance to Streptococcus pyogenes in the absence of detectable plasmid DNA (24). Although it is presumed that circular intermediates of conjugative transposons exist within the gram-positi...

Circularization of Tn916 is required for expression of the transposon‐encoded transfer functions: characterization of long tetracycline‐inducible transcripts reading through the attachment site

“…We have demonstrated previously that the presence of tetracycline in the mating medium increased the transfer frequency of Tn1545 (Doucet-Populaire et al, 1991). This Molecular Microbiology (1998) 28(1), [103][104][105][106][107][108][109][110][111][112][113][114][115][116][117] ᮊ 1998 Blackwell Science Ltd observation has been confirmed for several Tn916-like elements (Torres et al, 1991;Rice et al, 1992), including the prototype element Tn916 (Showsh and Andrew, 1992), and it has been proposed that tetracycline-stimulated excision of the element would result in an increase in excision frequency and, in turn, in an increase in conjugation frequency (Manganelli et al, 1995). The molecular basis for this hypothesis would be that the tetracycline-inducible tetM transcript might activate the transcription of the downstream genes xis-Tn and int-Tn (Rice et al, 1992).…”

“…This Molecular Microbiology (1998) 28(1), [103][104][105][106][107][108][109][110][111][112][113][114][115][116][117] ᮊ 1998 Blackwell Science Ltd observation has been confirmed for several Tn916-like elements (Torres et al, 1991;Rice et al, 1992), including the prototype element Tn916 (Showsh and Andrew, 1992), and it has been proposed that tetracycline-stimulated excision of the element would result in an increase in excision frequency and, in turn, in an increase in conjugation frequency (Manganelli et al, 1995). The molecular basis for this hypothesis would be that the tetracycline-inducible tetM transcript might activate the transcription of the downstream genes xis-Tn and int-Tn (Rice et al, 1992). The use of an excision-reporter plasmid to characterize the intracellular mobility of Tn916 in various Gram-positive bacteria led us to conclude that the excision event is not stimulated by the presence of tetracycline in the media (Celli et al, 1997).…”

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

“…However, data obtained by using non-quantitative PCR assays have led to a similar (Jaworski & Clewell, 1994) or to the opposite (Rice et al, 1992) conclusion. The excision frequency of pAT112, defined as the number of CmR excisants/total c.f.u., reflects the number of cells within a given bacterial population in which the amount of Xis-Tn and Int-Tn enabled an excision event to occur.…”

“…This observation was confirmed for several Tn916-like elements (Rice et al, 1995;Torres et al, 1991), including the prototype element Tn926 (Showsh & Andrew, 1992), and it has been proposed that tetracycline-stimulated excision of the element would result in an increase in excision frequency and, in turn, in an increase in conjugation frequency (Manganelli et al, 1995). The molecular basis for this hypothesis would be that the tetracycline-inducible tetM transcript might activate the transcription of the downstream genes xis-Tn and intTn (Rice et al, 1992). We do not agree with the hypothesis that tetracycline induces the excision of Tn926-like elements for the following reasons : (i) increased transcription of xis-Tn and int-Tn is not supposed to result in an increase of excision since this would not modify the ratio of the corresponding proteins; (ii) excision of Tn916 from the insertionally inactivated haemolytic gene carried by the IncHly plasmid pIP964, as measured by the reversion towards the haemolytic phenotype, did not increase when an E. faecalis host was cultivated in the presence of tetracycline (data not shown) ; (iii) the frequency of excision of pAT112 from pTCR9 was not increased when E .…”

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

“…Depending on the particular donor isolate, conjugation potentials can differ by 10,000-fold or more, yielding frequencies ranging from 10 Ϫ8 to 10 Ϫ4 per donor (21). Circular-intermediate DNA has been detected in extracts from E. faecalis cells with relatively high donor potentials (21,26) by using the PCR to amplify the region containing the two ends of the transposon joined together. Excised molecules were generally not detectable in the case of donors that transferred DNA at a very low frequency.…”