Transposase-dependent formation of circular IS256 derivatives in Staphylococcus epidermidis and Staphylococcus aureus

Abstract

IS256 is a highly active insertion sequence (IS) element of multiresistant staphylococci and enterococci. Here we show that, in a Staphylococcus epidermidis clinical isolate, as well as in recombinant Staphylococcus aureus and Escherichia coli carrying a single IS256 insertion on a plasmid, IS256 excises as an extrachromosomal circular DNA molecule. First, circles were identified that contained a complete copy of IS256. In this case, the sequence connecting the left and right ends of IS256 was derived from flanking DNA sequences of the parental genetic locus. Second, circle junctions were detected in which one end of IS256 was truncated. Nucleotide sequencing of circle junctions revealed that (i) either end of IS256 can attack the opposite terminus and (ii) the circle junctions vary significantly in size. Upon deletion of the IS256 open reading frame at the 3' end and site-directed mutageneses of the putative DDE motif, circular IS256 molecules were no longer detectable, which implicates the IS256-encoded transposase protein with the circularization of the element.

FIG. 1.

(A) IS256 and positions of outward-reading primers used for circle-specific PCRs. White boxes mark the left and right arms of the element containing the left (IR_L) and right (IR_R) inverted repeats. The gray rectangle represents the putative transposase gene (tnp256) of IS256. (B) Agarose gel electrophoresis of PCR fragments amplified with circle-specific outward-reading primers. Templates were obtained from extrachromosomal DNA preparations of S. epidermidis 307 (lane 1), S. epidermidis 307/95 (lane 2), E. coli(pIL2) (lane 3), E. coli(pIL2Δtnp) (lane 4), S. aureus(pIL2) (lane 5), S. aureus(pIL2Δtnp) (lane 6), S. aureus(pIL2-D167A) (lane 7), S. aureus(pIL2-D233A) (lane 8), and S. aureus(pIL2-E341A) (lane 9).

FIG. 2.

(A) Detection of IS256 circular forms by IS256-specific Southern hybridization of extrachromosomal DNA obtained from S. aureus(pIL2) (lanes 1 and 2) and S. aureus(pIL2Δtnp). (lanes 3 and 4). Lanes: 1, undigested plasmid fraction of pIL2; 2, ClaI-restricted plasmid fraction of pIL2; 3, undigested plasmid fraction of pIL2Δtnp; 4, ClaI-restricted plasmid fraction of pIL2Δtnp. The arrows mark the circular (lower) and linearized (upper) forms of the IS256 circles.(B) Rehybridization of the same blot with the vector backbone pRB472 but without the icaC:: IS256 insert.

FIG. 3.

Nucleotide sequence analyses of IS256 circle junctions detected in S. epidermidis, S. aureus, and E. coli. (A) Schematic representation of the IS256 insertion in the icaC gene in pIL2. The 8-bp target site duplications are marked by boldface letters. Dotted arrows indicate the putative strand transfer reactions of transposon ends into adjacent DNA. (B and C) Schematic illustration of IS256 circle junctions and nucleotide sequence analyses of complete (B) and truncated (C) circle junctions detected in S. epidermidis 307, S. epidermidis 307/95, E. coli(pIL2), and S. aureus(pIL2). IR_L, inverted repeat (left); IR_R, inverted repeat (right).