Table 1. MRSA252 uniquely shares multiple DNA sequences with other causative agents of bovine mastitis. Uniquely shared sequences Sequence description SAR1477 & SAR1478 SAR1562* SAR1892 & SAR1889 MRSA252 SAR0183 SAR0385 SAR0585 SAR0824 SAR0872* SAR1306 SAR1911 SAR2113 SAR2543 SAR2596 SAR2628 MRSA252 SAR0720 SAR0721 MRSA252 SAR0723 SAB1174 & SAB1175c SAB1330c & SAB1331c SAB1350c & SAB1349c SAB1663 & others RF122 SAB0122c SAB0361 SAB0449 SAB0723 SAB0770* SAB1691 SAB1884c SAB2336c SAB2390 SAB2423 FSL R2-503 FSL R2-503 SSPP217 Listeria monocytogenes S. saprophyticus PSSP2 plasmid Partial sequence of Acetylglutamate Kinase. Similar to bovine pathogenicity island protein ORF3 Hypothetical Phosphomethylpyrimidine kinase Malolactic enzyme ORF plus flanking sequences. Intragenic Hypothetical novel protein. Chorismate Synthase gene & Nucleoside Diphosphate Kinase Hypothetical novel proteins Hyaluronate lyase precursor 1 & other hypothetical proteins Hypothetical novel protein Hypothetical novel protein ATP binding ABC transporter Fructose 1,6 bisphosphatase Putative ATP-dependent protease ATP-binding subunit ClpL CopB (Copper cation ATPase transporter) Copper oxidase Cadmium-transporting ATPase Analysis of the MRSA252 Genome Flanking MGE or phage DNA None detected Phage None detected None detected None detected Transposon None detected Phage None detected None detected Phage None detected None detected None detected Transposon Transposon Transposon *Sequence flanks the gene. doi:10.1371/journal.pone.0003074.t001 Our comparative analysis identified additional putative HGT DNA sequences within the MRSA252 genome that have no counterpart in other Staphylococci examined in this study. For example, the unique DNA sequence found at nucleotides 303,347–306,097 contains an ORF coding for a 763 amino acid protein (Figure S1), annotated as a nitric oxide reductase (SAR0261) and identified as a region of inserted DNA [23]. A BLASTp database search revealed 51% amino acid identity to nitric oxide reductase encoding genes in Geobacillus species and Bacillus licheniformi (data not shown). In other bacterial pathogens, the nitric oxide reductase enzyme is considered a virulence factor that allows survival under very low oxygen tension and/or allows the organism to take advantage of de-nitrification to cope with nitric oxide production in macrophages [38,39]. The presence of mobile genetic element-associated genes integrated into the MRSA252 genome and the sharing of sequences between MRSA252 and RF122 prompted us to examine sequenced S. aureus associated MGEs for the presence of genes that might be shared by these two isolates, thus suggesting a potential mode of transfer. EvoPrinter analysis of the 13 different S. aureus isolates was performed using the 35 kb pTZ2162 plasmid as the reference input sequence. This plasmid is widely distributed among healthcare-associated MRSA strains [40]. The pTZ2162 EvoUnique profile identified multiple regions that are uniquely shared with different S. aureus genomes (data not shown). An EvoDifference analysis of one such region identified two flanking sub- regions, one shared with MRSA252 and the other with RF122 (Figure S2). The shared pTZ2162-MRSA252 sequence contains a partial sequence that matches the blaZ antibiotic resistance gene, which encodes ß-lactamase [41]. blaZ has been identified in a significant fraction of clinical S. aureus isolates [42] and in S. aureus isolates from persistent bovine mastitis [43]. The RF122 shared sequence contains an ORF that encodes a quinone oxidoreduc- tase/DT diaphorase, a member of a subfamily of alcohol dehydrogenases, annotated as SAB1296c [24]. In E. coli, oxidoreductases have been shown to be drug resistance factors [44]. The HGT events were most likely due to plasmid insertions, as both the MRSA252 (located at 866,929) and RF122 (located at 1,418,565 bp) sequences are flanked by plasmid sequences (data not shown). The pTZ2162 plasmid also shares near sequence identity to multiple genes in Staphylococcus haemolyticus and Staphylococcus epidermidis human pathogens (data not shown). EvoUnique profiles of other S. aureus isolates also identified putative HGT sequences that are not part of the MRSA252 or RF122 chromosomes. For example, our analysis revealed sequences within the human USA300 chromosome that are shared with S. epidermidis, S. haemolyticus and S. saprophyticus, but not with other S. aureus isolates (data not shown). Additionally, the USA300 EvoUnique profile revealed sequences uniquely shared with the human MW2 and Col isolates, but no sequences were found uniquely shared between USA300 and MRSA252 or RF122. Summary The finding of multiple putative HGT DNAs that are shared between a human epidemic MRSA isolate and a contagious bovine mastitis S. aureus isolate, but that are absent from other S. aureus, indicates that these two phylogenetically distinct strains most likely have undergone multiple gene transfers either between themselves or with as yet unidentified additional bacteria. Clearly, comparisons between bacterial genomes establish only that they uniquely share DNA sequences, but the analysis does not establish a transfer mechanism or the identity of the exchanging partners. PLoS ONE | www.plosone.org 4 August 2008 | Volume 3 | Issue 8 | e3074