Tn5386, a novel Tn916-like mobile element in Enterococcus faecium D344R that interacts with Tn916 to yield a large genomic deletion - PubMed (original) (raw)

Tn5386, a novel Tn916-like mobile element in Enterococcus faecium D344R that interacts with Tn916 to yield a large genomic deletion

Louis B Rice et al. J Bacteriol. 2005 Oct.

Abstract

We describe Tn5386, a novel ca.-29-kb Tn916-like mobile element discovered to occur in ampicillin-resistant, Tn916-containing Enterococcus faecium D344R. PCR amplification experiments after overnight growth with or without tetracycline revealed "joint" regions of circularized Tn5386 composed of 6-bp sequences linking different transposon termini. In one case (no tetracycline), the termini were consistent with those derived by target site analysis of the integrated element. In the other case, the termini were virtually identical in distance from the integrase binding regions, as seen with Tn916. These data are consistent with a model in which one PCR product results from the action of Tn5386 integrase, whereas the other results from the action of the Tn916 integrase on Tn5386. Spontaneous conversion of D344R to an ampicillin-susceptible phenotype (D344SRF) was associated with a 178-kb deletion extending from the left end of Tn5386 to the left end of Tn916. Examination of the Tn5386 junction after the large deletion event suggests that the deletion resulted from an interaction between the nonintegrase ends of Tn5386 and Tn916. The terminus of Tn5386 identified in this reaction suggested that it may have resulted from the activity of the Tn916 integrase (Int(Tn916)). The "joint" of the circular element resulting from this excision was amplifiable from D344R, the sequence of which revealed a heteroduplex consistent with Int(Tn916)-mediated excision. In contrast, Tn5386 joints amplified from ampicillin-susceptible D344SRF revealed ends consistent with Tn5386 integrase activity, reflecting the absence of Tn916 from this strain. Tn5386 represents a new member of the Tn916 transposon family. Our data suggest that excision of Tn5386 can be catalyzed by the Tn916 integrase and that large genomic deletions may result from the interaction between these heterologous elements.

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Figures

FIG. 1.

Proposed map of E. faecium D344R and D344S in the areas of the excised segment. The positions of Tn_916_, Tn_5386_, pbp5, and the SmaI site downstream of pbp5 are marked. The large plasmid clones containing either Tn_916_ or Tn_5386_ from the different strains are marked by lines terminating in filled circles. The regions referred to in the text as left and right junctions of the two mobile elements are marked above the transposons. Arrows above marked genes represent direction of transcription. Regions for which probes were generated for use in the Southern hybridization in Fig. 2 are marked by single and double asterisks. pbp5, penicillin binding protein 5 gene; int, integrase genes.

FIG. 2.

Pulsed-field gel electrophoresis of SmaI-digested genomic DNA from E. faecium D344R and E. faecium D344SRF. Lane 1, bacteriophage lambda concatemer size standard (sizes are marked to the left); lane 2, D344R digested with SmaI; lane 3, D344SRF digested with SmaI; lanes 4 and 5, SmaI digestions seen in lanes 2 and 3 hybridized with a probe derived from the left flanking region (depicted as single asterisk in Fig. 1); and lanes 6 and 7, SmaI digestions seen in lanes 2 and 3 hybridized with a probe derived from the right flanking region (depicted as double asterisk in Fig. 1). Note that the left and right flanking region probes hybridize with the same fragment in D344SRF, consistent with excision of the region between the left flanking region and Tn_5386_. The lower hybridizing band in D344R likely represents a doublet in which only one of the bands hybridizes to the probe.

FIG. 3.

Comparison of nucleotides composing the termini of Tn_916_ and Tn_5386_. (A) Comparison of the termini at the nonintegrase ends of the transposons. The DR2 integrase binding sites are underlined, and identical nucleotides are marked by vertical lines. The boxed region is the coupling sequence that lies adjacent to the end of Tn_5386_ in D344R as defined by analysis of the target sites and the circular form resulting in the larger joint PCR product. The arrow reflects the end of Tn_5386_ as defined in the circular form resulting in the smaller PCR product (presumed to result from the action of IntTn_916_ on the ends of Tn_5386_). This also reflects the end of the transposon used to yield the excision of the larger region resulting in the creation of D344S. (B) Comparison of the termini at the integrase ends of the transposons. The DR2 integrase binding sites are underlined, and identical nucleotides are marked by vertical lines. The arrow reflects the end of Tn_5386_ as defined in the circular form resulting in the smaller PCR product (presumed to result from the action of IntTn_916_ on the ends of Tn_5386_). Extending beyond the arrow is the Tn_5386_ terminus as defined by analysis of the target sites and the circular form resulting in the larger joint PCR product.

FIG. 4.

Comparison of left and right Tn_5386_ junction sequences with the target sequence as defined by the E. faecium database and the D344R genome after Tn_5386_ excision. The ends of Tn_5386_ are marked by italics. The coupling sequence brought with it by Tn_5386_ is marked by the solid box adjacent to the left end of Tn_5386_. The coupling sequence representing the target is marked by the 6-base-pair sequence in the hatched box adjacent to the right end of Tn_5386_.

FIG. 5.

PCR amplification of the Tn_5386_ joint after growth of D344R with tetracycline (lane B) and without tetracycline (lane C). Size markers are shown at the left. Identical primers (primers 101 and 102) were used to generate the products in both instances. A small amount of the smaller product is visible in lane B (arrow), but the larger product is absent from lane C.

FIG. 6.

Tn_5386_ joints and regenerated target sequences generated from amplification performed on DNA extracted from D344R grown in the presence of tetracycline (10 μg/ml). In four of four (4/4) clones, the joint sequence of the circular form was identical to the coupling sequence (TATCAC) found at the left junction of Tn_5386_. Similarly, in four of four clones the coupling sequence (CATGTT) found at the right junction of Tn_5386_ is present in the regenerated target sequence.

FIG. 7.

Comparison of left junction of Tn_916_ in D344R with the left junctions of Tn_5386_ in D344R and D344SRF. (A) Left Tn_916_ junction within the D344R genome. The genome sequence is presented in lowercase, while the transposon sequence is in italics. The hatched box represents the 6-bp coupling sequence between Tn_916_ and the flanking sequence. (B) Left junction sequence of Tn_5386_ in D344R. As above, the genomic sequence is in lowercase, and the transposon is italicized. The 6-bp coupling sequence is boxed. (C) Left junction sequence of Tn_5386_ in D344SRF, after excision of the pbp5 region. The putative new coupling sequence is now in the hatched and dotted box, and the “new” end of Tn_5386_ is incorporated into the italicized sequence representing the transposon.

FIG. 8.

Proposed mechanism for excision of the large pbp5_-containing region from D344R. The orientation of the two transposons in D344R and the junction sequences are depicted at the top of the figure (A). We propose that the transposons align as depicted in panel B, with strand exchange occurring between the regions denoted by the X. This yields two products: a circularized form that contains both pbp5 and Tn_916 (C) and a regenerated chromosomal region that contains Tn_5386_ linked to the region that flanked Tn_916_ in D344R (D). The asterisks denote regions of newly joined DNA. Below the circularized form in panel C are depicted the two different joints found in the PCR products, with their relative prevalences among the four inserts sequenced. i, integrase.

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Tn5386, a novel Tn916-like mobile element in Enterococcus faecium D344R that interacts with Tn916 to yield a large genomic deletion - PubMed (original) (raw)