<p>1Supplementary data</p><p>2</p><p>1 1 3Figure S1. Phylogenetic tree of fosfomycin resistance proteins. Sequences were aligned </p><p>4by ClustalW, and a dendrogram was generated using the neighbor-joining method. </p><p>5Bootstrap values (n=10,000) are shown for the major nodes. The bacterial source and the </p><p>6GenBank accession number are given for each FOS protein. The three classes of </p><p>7fosfomycin resistance proteins are indicated: FosA (see references in text), Fos B (Zilhao,</p><p>8R., Courvalin, P. (1990) Nucleotide sequence of the fosB gene conferring fosfomycin </p><p>9resistance in Staphylococcus epidermidis. FEMS Microbiol. Lett 68, 267-272. and Cao, </p><p>10M., Bernat, B.A., Wang, Z., Armstrong R.N., Helmann, J.D. (2001) FosB, a cysteine-</p><p>11dependent fosfomycin resistance protein under the control of σw, an extracytoplasmic-</p><p>12function σ factor in Bacillus subtilis. J. Bacteriol 183, 2380-2383.) and FosX (Fillgrove, </p><p>13K.L., Pakhomova, S., Newcomer, M.E., Armstrong, R.N. (2003) Mechanistic diversity of </p><p>14fosfomycin resistance in pathogenic microorganisms. J. Am. Chem. Soc 125, 15730-</p><p>1515731.).</p><p>16</p><p>2 2 17</p><p>18 19 20Figure S2. Location of fosA2 gene identified by pulsed field gel electrophoresis. Total </p><p>21genomic DNA of Sam066F1 was digested with I-CeuI nuclease (“a” lanes) or S1 </p><p>22nuclease (“b” lanes) and probed with a labeled 23S rRNA gene (lanes a2, a3) or the </p><p>23712bp fosA2 fragment (lane b1, b2). Lane M contains a λ concatamer size ladder (New </p><p>24England Biolabs). Arrows indicate fosA2 hybridization. DNA was prepared and digested </p><p>25as described by the method of Barton et al. (Barton, B.M., Harding, G.P., Zuccarelli, A.J. </p><p>26(1995) General method for detecting and sizing large plasmids Anal. Biochem 226, 235-</p><p>27240.) and Liu et al. (Liu, S.L., Hessel, A., Sanderson, K.E. (1993) Genomic mapping with</p><p>28I-Ceu I, an intron-encoded endonuclease specific for genes for ribosomal RNA, in </p><p>29Salmonella spp., Escherichia coli, and other bacteria. Proc Natl Acad Sci USA 90, 6874–</p><p>306878.). The 23S RNA gene probe was synthesized by PCR using primer pair 23S-43af </p><p>3 3 31(5'-GGATGTTGGCTTAGAAGCAG-3') and 23S-62ar (5'-</p><p>32CTTAGGACCGTTATAGTTAC-3') on genomic DNA of Sam066F1. Both probes were </p><p>33synthesized using the (DIG High Prime DNA Labeling and Detection Starter KitI, </p><p>34Roche). Digested DNA was subjected electrophoresis at 5 to 120 seconds pulse time for </p><p>3548 hours in 1% agarose gel in TBE in a BIO-RAD CHEF MAPPER XA (BioRad) and </p><p>36transferred to Hybond-N+ nylon membrane (Amersham Biosicences) for hybridization </p><p>37and washing according to manufacturer’s instructions (DIG High Prime DNA Labeling </p><p>38and Detection Starter KitI, Roche). </p><p>39</p><p>40</p><p>41</p><p>4 4</p>