as implemented in our software package. The maximal number of top level nodes was set to 2. As mentioned in the Figure legend 1, the arrangement of spectra on the left site of the dendrogram is arbitrary. Species with distance levels under 500 are reliably classified. DNA-based phylogenetic analysis (Figure 2) was done using the Mega (Molecular Evolutionary Genetics Analysis) program, version 3.1 (http://www.megasoftware.net/) [15]. The complete set of reference spectra compiled in the database of our software package is linked to the NCBI taxonomy database (http://www.ncbi.nlm.nih.gov/Taxonomy/). For identity scoring, the algorithm implemented in the Biotyper software counted mass signals in experimental spectra that matched with reference spectra and vice versa. Furthermore, the algorithm applied correlates signal intensities of matched signals. Together, three scores obtained from such a procedure are multiplied and normalized to a value of 1000 and then converted in its common logarithm (3). Log scores over 2 indicated a reliable identification of species; log scores over 1.7 generally meant a reliable identification of bacterial genera. Log scores of 3 were obtained when spectra matched with themselves. For the identification of bacterial species, this pattern matching algorithm was routinely applied. For the distinction of highly similar mass spectra of closely related sub species, we used a weighted pattern matching algorithm. In practice, we assigned additional values to informative mass signals that were found in the reference spectra of these sub species. For the application of weighted pattern matching we used the masses and settings listed in Figure 5 and in Table 3. For more details on the BioTyper software the reader is referred to a handbook that is available from the authors as a hardcopy or an electronic version (CD of the complete analysis software package that is freely available for reproducing the results of this study and for testing the procedure shown in this article for additional bacterial genera). SNP genotyping: Approximately 5 mg of bacterial pellet was re- suspended in 1 ml 0.1% Tween-20 and heated up to 65uC for References 1. Sintchenko V, Iredell JR, Gilbert GL (2007) Pathogen profiling for disease management and surveillance. Nat Rev Microbiol 5: 464–70. 2. Enright MC, Spratt BG (1999) Multilocus sequence typing. Trends Microbiol 7: 482–487. 3. Jock S, Geider K (2004) Molecular differentiation of Erwinia amylovora strains from North America and of two Asian pear pathogens by analyses of PFGE patterns and hrpN genes. Environ Microbiol 6: 480–90. 4. Sampath R, et al. (2007) Global surveillance of emerging Influenza virus genotypes by mass spectrometry. PLoS ONE 2: e489. 5. Honisch C, et al. (2007) Automated comparative sequence analysis by base- specific cleavage and mass spectrometry for nucleic acid-based microbial typing. Proc Natl Acad Sci U S A 104: 10649–10654. 6. Claydon MA, Davey SN, Edwards-Jones V, Gordon DB (1996) The rapid identification of intact microorganisms using mass spectrometry. Nat Biotechnol 14: 1584–1586. 7. Holland RD, et al. (1996) Rapid identification of intact whole bacteria based on spectral patterns using matrix-assisted laser desorption/ionization with time-of- flight mass spectrometry. Rapid Commun Mass Spectrom 10: 1227–1232. 8. Chong BE, Wall DB, Lubman DM, Flynn SJ (1997) Rapid profiling of E. coli proteins up to 500 kDa from whole cell lysates using matrix-assisted laser Bacteria/Mass Spectrometry 15 min. One micoliter was used as template for subsequent PCR. PCR was carried out in 10 ml volume. The PCR buffer consisted of 20 mM (NH4)2SO4, 75 mM Tris-HCl (pH 9.0), 0.01% Tween- 20, 2.5 mM MgCl2, 0.5 M betaine solution, 0.3 mM dNTPs, 1 U conventional Taq polymerase (produced in-house), 0.025 U proofreading Taq polymerase (produced in-house), 0.3 mM forward primer (59-CGATGACGTGGTGATACTGG-39), 0.3 mM reverse primer (59-TCGACTCCCCTACAGCCTTA- 39). After denaturating the PCR samples for 5 minutes at 95uC, amplifications were carried out at 94uC for 30 seconds, 65uC for 30 seconds, and 72uC for 30 seconds for 35 cycles. Finally, the samples were incubated at 72uC for 5 minutes. SNP genotypes were detected by mass spectrometry with the standard GOOD assay in negative ion mode [13,16] as described in full detail in ref. 13 by using 2 ml of the PCR products generated from 10 ml reactions. The extension primer used for the GOOD assay was 59- GCGACTTTCTTCGAAGGGG*AC-39 (* indicates a phosphor- othioate linkage). The reference sequences of the galE gene of two E. amylovora strains are shown in Figure S1. Supporting Information Figure S1 Reference sequences. A SNP in the gal-E gene was used to differentiate E. amylovora strains of European/Mediter- ranean (Ea1/79) from American origin (Ea273). Found at: doi:10.1371/journal.pone.0002843.s001 (0.02 MB DOC) Author Contributions Conceived and designed the experiments: SS MK KG. Performed the experiments: SS AF TM KG. Analyzed the data: SS AF TM. Contributed reagents/materials/analysis tools: SS MK RR MK KG. Wrote the paper: SS AF MK KG. desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 11: 1900–1908. 9. Starr MP, Chatterjee AK (1972) The genus Erwinia: enterobacteria pathogenic to plants and animals. Annu Rev Microbiol 26: 389–426. 10. Crosse JE, Bennett M, Garett CME (1958) Nature 182: 1530. 11. Fenselau C, Demirev PA (2001) Characterization of intact microorganisms by MALDI mass spectrometry. Mass Spectrom Rev 20: 157–171. 12. Kim WS, Gardan L, Rhim SL, Geider K (1999) Erwinia pyrifoliae sp. nov., a novel pathogen that affects Asian pear trees (Pyrus pyrifolia Nakai) Int J Syst Bacteriol 49: 899–905. 13. Sauer S, Reinhardt R, Lehrach H, Gut IG (2006) Single-nucleotide polymorphisms: analysis by mass spectrometry. Nat Protoc 1: 1761–1771. 14. Sauer S, et al. (2005) Miniaturization in functional genomics and proteomics. Nat Rev Genet 6: 465–476. 15. Jukes TH, Cantor CR in Munro HN, ed (1969) Mammalian protein metabolism. New York: Academic Press. pp 21–123. 16. Sauer S (2006) Typing of single nucleotide polymorphisms by MALDI mass spectrometry: principles and diagnostic applications. Clin Chim Acta. 363: 95–105. PLoS ONE | www.plosone.org 10 July 2008 | Volume 3 | Issue 7 | e2843