Gene acquisition at the insertion site for SCCmec, the genomic island conferring methicillin resistance in Staphylococcus aureus

Abstract

Staphylococcus aureus becomes resistant to methicillin by acquiring a genomic island, known as staphylococcal chromosome cassette mec (SCCmec), which contains the methicillin resistance determinant, mecA. SCCmec is site-specifically integrated into the staphylococcal chromosome at a locus known as the SCCmec attachment site (attB). In an effort to gain a better understanding of the potential that methicillin-sensitive S. aureus (MSSA) isolates have for acquiring SCCmec, the nucleotide sequences of attB and surrounding DNA regions were examined in a diverse collection of 42 MSSA isolates. The chromosomal region surrounding attB varied among the isolates studied and appears to be a common insertion point for acquired foreign DNA. Insertions of up to 15.1 kb were found containing open reading frames with homology to enterotoxin genes, restriction-modification systems, transposases, and several sequences that have not been previously described in staphylococci. Two groups, containing eight and four isolates, had sequences found in known SCCmec elements, suggesting SCCmec elements may have evolved through repeated DNA insertions at this locus. In addition, the attB sequences of the majority of MSSA isolates in this collection differ from the attB sequences of strains for which integrase-mediated SCCmec insertion or excision has been demonstrated, suggesting that some S. aureus isolates may lack the ability to site-specifically integrate SCCmec into their chromosomes.

FIG. 1.

eBURST diagram of all S. aureus isolates examined in this investigation. The interrelatedness, based on multilocus sequence typing, of all isolates in this investigation is shown as calculated using eBURST with the default settings. The dot size is representative of the number of isolates with a given ST. Any relationship between STs is indicated by a solid line for double-locus variants and a dotted line for single-locus variants. Only five related isolate groups were present in this study; ST8 and ST195 (group 1), ST9 and ST109 (group 2), ST 45 and ST47 (group 3), ST1 and ST188 (group 4), and ST5 and ST6. All other isolates are unrelated.

FIG. 2.

Site-specific chromosomal integration and excision of SCCmec in S. aureus 450 M. S. aureus 450 M is an 8325 derivative containing SCCmec. Schematics of the 450 M chromosome with SCCmec integrated or excised are shown at the top, with triangles indicating primer binding sites, numbers indicating the region amplified in the corresponding lane of the gel, and vertical lines indicating attachment sites. Select open reading frames are indicated as reference points. Shown in lanes 1 and 2 are PCR amplification of the right and left SCCmec attachment sites using primers I1F/colA and I1R/colB, respectively, indicating that SCCmec is site-specifically integrated into the chromosome of 450 M. Shown in lane 3 is PCR amplification of the chromosomal junction from which SCCmec was site-specifically excised by CcrAB, using primers I1F and I1R. Lane 4 contains the PCR amplification product of the excised, circular SCCmec element using primers colA and colB.

FIG. 3.

Schematic of the genetic regions surrounding attB in seven MSSA isolates. The nucleotide sequences surrounding attB in strains 8325, 15585, 15666, 15653, 3298, 15604, and 15584 are depicted. Block arrows represent predicted open reading frames, and their fill patterns indicate their putative functions, as shown in the key. Where possible, the predicted function was assigned based on homology. Open reading frames with similar predicted functions are not identical in the different isolates. Each region is flanked by orfX (gray arrow) and a conserved predicted open reading frame (white arrow). 15666 and 15653 differ only by the insertion of a putative transposase into 15653 that is not present in 15666. 3298 and 15604 share a region at the left end that encodes a putative enterotoxin (boxed in gray). Open reading frames labeled hsdS, hsdR, and hsdM are predicted components of a type 1 restriction-modification system.

FIG. 4.

Comparison of the chromosomal regions of MRSA252, 15580, NRS199, and 15682, as well as MW2 and 15575. (A) The chromosomal region containing SCCmec and the surrounding sequence of MRSA252 is shown, along with similar regions from MSSA strains 15580, NRS199, and 15682. MRSA252 contains SCCmec type II and a 6-kb region encoding two regions homologous to transposases inserted into attB. The left end of this chromosomal region contains two open reading frames with homology to restriction and modification genes. Strain 15580 differs from MRSA252 by the absence of SCCmec. Strains NRS199 and 15682 contain the two open reading frames with homology to restriction and modification genes, but the other regions are absent in these strains. (B) The chromosomal region containing SCCmec and surrounding sequence in MW2 is shown, along with a similar region from MSSA 15575. MW2 carries SCCmec type IV. The region outside of SCCmec contains an open reading frame also present within the right end of SCCmec (MW0048 and MW0025, respectively). This region also contains a putative transposase, staphylococcal enterotoxin H (seh), and a truncated region with homology to staphylococcal enterotoxin O (seo). 15575 differs from MW2 by the absence of SCCmec type IV. Each region is flanked by orfX (gray arrow) and a conserved predicted open reading (white arrow). Brackets are used to show the sequence difference between strains. SCCmec elements are not drawn to scale.

FIG. 5.

Nucleotide alignment of attB genes and surrounding regions from 18 S. aureus strains. The thick black arrow indicates the portion of the orfX coding region shown. The thin black arrow marks the 15-bp core sequence of the SCCmec attachment site. This sequence is present at the carboxyl terminus orfX and, when SCCmec inserts, is directly repeated at the other end of the element. SCCmec and its point of chromosomal insertion are depicted above the alignment (not drawn to scale). The region shaded in light gray represents a conserved sequence present outside of attB in all S. aureus strains known to undergo CcrAB-mediated SCCmec integration or excision. The conserved sequence of orfX is shaded in black, while strains are indicated on the left. The attB region of strain MW2 was inferred based on the genome sequences of attL and attR, since SCCmec did not excise in MW2.

FIG. 6.

DNA acquisition and deletion within SCCmec. A schematic of a generic SCCmec element is shown at the top, with block arrows representing selected open reading frames and flags indicating the left and right SCCmec attachment sites. The sequences of SCCmec types IV, IVa, IVb, IVc, IVe, IVg, and I are depicted in cartoon fashion (GenBank accession numbers NC_003923, AB063172, AB063173, AB096217, AJ810121, DQ106887, and NC_002951, respectively). Regions of the SCCmec elements with >90% nucleotide identity are filled with the same color/pattern, while different fill patterns indicate entirely different blocks of sequence. Each of these elements contains a core sequence including IS431, mecA, and a truncated mecRI (shown in gray). All SCCmec type IV elements share a region encoding ccrA2B2 (checkered), while SCCmec type I contains a homologous, yet distinct, region encoding ccrA1B1 (cross-hatched). The left and right ends of SCCmec vary among types and subtypes in a manner consistent with the insertion or deletion of large blocks of nonhomologous sequence.