Analysis of the MRSA252 Genome Figure 1. Circular displays of the S. aureus human MRSA252 and bovine RF122 chromosomes showing the relative genomic positions of the uniquely shared DNAs. Shown are the relative positions of the MRSA252 (SAR) and RF122 (SAB) shared genes. The chromosomal position of the MRSA252 - Listeria monocytogenes shared genes copper ATPase and copper oxidase is indicated in green. The chromosome position of the cadmium-transporting ATPase cadA gene shared with the S. saprophyticus pSSP2 plasmid is indicated in orange. Arrows indicate orthologous DNAs. doi:10.1371/journal.pone.0003074.g001 greater identity with the closely-related genomes (that lack the unique DNA) than they do with the putative donor genome. For example, pairwise alignments reveal that the highest shared identity with 5 kb of MRSA252 DNA flanking either side of the malolactic enzyme gene (Figure 2) is not with RF122 but with the human community-acquired MRSA MW2 isolate (data not shown). Of all of the genomes included in the analysis, the RF122 flanking DNA is least homologous to the MRSA252 flanks. The multiplicity and synonymous genomic locations of many of the unique DNAs indicate that multiple HGT events have occurred, and that homologous recombination most likely played a role in many of the integration events. The bovine RF122 isolate or related clones may have undergone HGT events with other human S. aureus isolates. For example, DNA sequences between bases 404,577 and 409,784 of RF122 are exclusively shared with two human isolates, MSSA476 and MW2, but not with MRSA252 or the other S. aureus isolates examined. MSSA476 and MW2 are representative of community-acquired S. aureus strains; MSSA476 is a hyper-virulent community-acquired methicillin-susceptible strain isolated in the United Kingdom [23] and the MW2 strain is one of the major MRSA pathogens causing community-acquired infections in the mid-western region of the USA [31]. The shared sequences contain ORFs but do not encode proteins of identifiable function (data not shown). EvoPrinter analysis of the MRSA252 chromosome also identified additional DNA sequence blocks that are absent from the other S. aureus but shared with other causative agents of bovine mastitis: Listeria monocytogenes and S. saprophyticus [32,33]. Most notable is a region (at position 754,883) that spans the copper cation ATPase transporter copB (SAR0720) and copper oxidase genes (SAR0721) (Figure 1 and 3A). The MRSA252 genomic region that spans these two genes is nearly identical to DNA present in the genome of Listeria monocytogenes, as revealed by a BLASTn alignment (data not shown). Pairwise alignments between the Listeria DNA and the aligning region of the MRSA252 chromosome reveal that the two DNAs share 99.8% identity over 3,131 bp (Figure 3A). Our analysis also uncovered another human MRSA isolate, USA300, that contains a copper ATPase transporter gene (SAUSA300_ 0078, identified as copA), however, its sequence homology to the MRSA252/Listeria genes is significantly less and it lacks the flanking copper oxidase gene (Figure 3B). The MRSA252 copB and copper oxidase genes were most likely transferred via a plasmid, as the flanking DNA in MRSA252 is homologous to plasmid sequences (data not shown). The copA and copper oxidase genes constitute a copper resistance operon (cop) associated with resistance to metal toxicity [34]. It is surprising that Listeria and MRSA252 have near identical copper resistance genes. Metal ion transporters play important roles in both nutrient uptake and in secretion of toxins [35]. Another MRSA252 unique sequence block (2,152 bp; SAR0723) is also shared with the Staphylococcus saprophyticus plasmid pSSP2 (locus tag: SSPP217, GenBank accession NC_007352) and it encodes the cadmium-transporting ATPase CadA protein (93.5% shared identity). Heavy metal cadmium resistance has been associated with S. aureus plasmids [36], although the current selective pressure for cadmium resistance is unknown [37]. PLoS ONE | www.plosone.org 3 August 2008 | Volume 3 | Issue 8 | e3074