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Comparative Study
. 2010 Sep;65(9):1894-906.
doi: 10.1093/jac/dkq219. Epub 2010 Jun 17.

Tn1546 Is Part of a Larger Plasmid-Encoded Genetic Unit Horizontally Disseminated Among Clonal Enterococcus Faecium Lineages

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Free PMC article
Comparative Study

Tn1546 Is Part of a Larger Plasmid-Encoded Genetic Unit Horizontally Disseminated Among Clonal Enterococcus Faecium Lineages

H Sletvold et al. J Antimicrob Chemother. .
Free PMC article

Abstract

Objectives: To determine the genetic composition of the first VanA-type plasmid (pIP816) reported, which was isolated from a clinical Enterococcus faecium (BM4147) strain in France in 1986, and to reveal the genetic units responsible for the dissemination of the vanA gene cluster by comparisons with current, published and additionally generated vanA-spanning plasmid sequences obtained from a heterogeneous E. faecium strain collection (n = 28).

Methods: Plasmid sequences were produced by shotgun sequencing using ABI dye chemistry and primer walking, and were subsequently annotated. Comparative sequence analysis of the vanA region was done with published plasmids, with a partial vanA plasmid (pVEF4) reported here and to >140 kb of sequence obtained from a collection of vanA-harbouring plasmid fragments.

Results: Bioinformatic analyses revealed that pIP816 from 1986 and contemporary vanA plasmids shared a conserved genetic fragment of 25 kb, spanning the 10.85 kb vanA cluster encoded by Tn1546, and that the larger unit is present in both clinical and animal complexes of E. faecium. A new group II intron in pVEF4 was characterized.

Conclusions: Comparative DNA analyses suggest that Tn1546 disseminates in and between clonal complexes of E. faecium as part of a larger genetic unit, possibly as a composite transposon flanked by IS1216 elements.

Figures

Figure 1
Figure 1
Genetic map of pIP816 and pVEF4. Coding regions are represented by arrows indicating the direction of transcription and are coloured according to their predicted functions. The inverted repeats (IR) of the Tn1546 transposon and the predicted origin of replication (oriR) of the plasmids are given as black boxes. The group II intron En.fm.I2 of pVEF4 is shown as dark grey boxes flanking the intron-encoding protein. Thin arrows indicate the 25 kb larger genetic unit. Truncated CDSs are indicated with a prime symbol (e.g. tnp′).
Figure 2
Figure 2
Gene organization of Tn1546 and flanking areas in vanA plasmids from genomically different E. faecium strains of human or animal origin. Identical coding regions are colour-coded to highlight similarities in the Tn1546 flanking regions. Similar colour indicates identity. White, Tn1546; red, IS1216; dark grey, intron En.fm.I2 and iep. Note that the ∼7 kbp region flanked by IS1216 (red arrows) in pVEF1–pVEF3 is inverted in pIP816, and that similar organization was found in plasmids from two GREF from two Norwegian poultry farms (strains 399/F99/A8 and 64/F98/A1). The top line indicates the size of the aligned Tn1546 flanking regions, with positional marks in kbp.
Figure 3
Figure 3
Structural features of En.fm.I2 and its intron-encoded protein (IEP). (a) Predicted secondary RNA structure of En.fm.I2. Intron nucleotides are written in capital letters and exon sequences are written in lowercase letters. Roman numerals denote the domains I–VI. The IEP is found in domain IV. Intron-binding sites (IBSs) 1 and 2 along with the exon-binding sites (EBSs) 1 and 2 are marked by arrows and boxes, respectively. IBS/EBS3 is a single nucleotide interaction and denoted by pointing arrows. The bulged A (branch site) is located in domain VI and shown in bold. (b) The putative IEP displays a reverse transcriptase (RT) domain (bold letters), a maturase (X) domain (italics) and an endonuclease (En) domain (grey). All introns analysed, except two, had identical amino acid composition to En.fm.I2 of pVEF4 (no. 54, top). The non-synonymous substitutions in En.fm.I2 from E. faecium strains 31/F01/H (no. 49) and TUH32-79 (no. 45) are shown in the alignment (identical amino acids are represented by a dot; a dash indicates gaps or substitutions).
Figure 4
Figure 4
RT–PCR analyses of the topoisomerase, intron, intron splicing products and enterococcal elongation factor. RT–PCR products from pVEF4 and pVEF3 are given on alternate lanes 1–10. PCR products are shown as follows: topo mRNA without intron (lanes 1 and 2, primer pair ip3F/giiR7); 5′ intron–exon junction (lanes 3 and 4, primer pair ip3F/giiR8); 3′ intron–exon junction (lanes 5 and 6, primer pair giiF5/giiR7); intron lariat structure (lanes 7 and 8, primer pair giiF5/giiR8); and positive RT–PCR control (lanes 9 and 10, primer pair Ent1/Ent2). Ladder (lanes L), 100 bp DNA molecular size marker from New England Biolabs. The sequence data of the ligated exon with the indicated splice site is shown in the lower half of the figure.

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