nigan, San Jose, CA; version v.27, rev. 11). Sequest was searched with a fragment ion mass tolerance of 1.00 Da and a parent ion tolerance of 10 ppm. Up to two missed tryptic cleavages were allowed. Methionine oxidation (+15.99492 Da), Carbamido- methylation (+57.021465 Da) and phosphorylation of STY (+79.966331 Da) was set as variable modification. Proteins were identified by at least two peptides applying a stringent SEQUEST filter. Sequest identifications required at least deltaCn scores of greater than 0.10 and XCorr scores of greater than 1.9, 2.2, 3.75 and 3.75 for singly, doubly, triply and quadruply charged peptides. Phosphorylated peptides which passed this filter were examined manually and accepted only, when b- or y- ions confirmed the phosphorylation site. Supporting Information Table S1 PknB-phosphorylated peptides Found at: doi:10.1371/journal.pone.0009057.s001 (0.15 DOC) MB References 1. Rosqvist R, Forsberg A, Wolf-Watz H (1991) Microinjection of the Yersinia YopE cytotoxin in mammalian cells induces actin microfilament disruption. Biochem Soc Trans 19: 1131–1132. 2. Rosqvist R, Magnusson KE, Wolf-Watz H (1994) Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells. EMBO J 13: 964–972. 3. Galyov EE, Hakansson S, Forsberg A, Wolf-Watz H (1993) A secreted protein kinase of Yersinia pseudotuberculosis is an indispensable virulence determinant. Nature 361: 730–732. 4. Juris SJ, Rudolph AE, Huddler D, Orth K, Dixon JE (2000) A distinctive role for the Yersinia protein kinase: actin binding, kinase activation, and cytoskeleton disruption. Proc Natl Acad Sci U S A 97: 9431–9436. 5. Wiley DJ, Shrestha N, Yang J, Atis N, Dayton K, et al. (2009) The activities of the Yersinia protein kinase A (YpkA) and outer protein J (YopJ) virulence factors converge on an eIF2alpha kinase. J Biol Chem 284: 24744–24753. 6. Navarro L, Koller A, Nordfelth R, Wolf-Watz H, Taylor S, et al. (2007) Identification of a molecular target for the Yersinia protein kinase A. Mol Cell 26: 465–477. 7. Munoz-Dorado J, Inouye S, Inouye M (1993) Eukaryotic-like protein serine/ threonine kinases in Myxococcus xanthus, a developmental bacterium exhibiting social behavior. J Cell Biochem 51: 29–33. 8. Perez J, Castaneda-Garcia A, Jenke-Kodama H, Muller R, Munoz-Dorado J (2008) Eukaryotic-like protein kinases in the prokaryotes and the myxobacterial kinome. Proc Natl Acad Sci U S A 105: 15950–15955. 9. Av-Gay Y, Everett M (2000) The eukaryotic-like Ser/Thr protein kinases of Mycobacterium tuberculosis. Trends Microbiol 8: 238–244. 10. Wang J, Li C, Yang H, Mushegian A, Jin S (1998) A novel serine/threonine protein kinase homologue of Pseudomonas aeruginosa is specifically inducible within the host infection site and is required for full virulence in neutropenic mice. J Bacteriol 180: 6764–6768. 11. Motley ST, Lory S (1999) Functional characterization of a serine/threonine protein kinase of Pseudomonas aeruginosa. Infect Immun 67: 5386–5394. 12. Pallova P, Hercik K, Saskova L, Novakova L, Branny P (2007) A eukaryotic-type serine/threonine protein kinase StkP of Streptococcus pneumoniae acts as a dimer in vivo. Biochem Biophys Res Commun 355: 526–530. 13. Echenique J, Kadioglu A, Romao S, Andrew PW, Trombe MC (2004) Protein serine/threonine kinase StkP positively controls virulence and competence in Streptococcus pneumoniae. Infect Immun 72: 2434–2437. 14. Novakova L, Saskova L, Pallova P, Janecek J, Novotna J, et al. (2005) Characterization of a eukaryotic type serine/threonine protein kinase and protein phosphatase of Streptococcus pneumoniae and identification of kinase substrates. FEBS J 272: 1243–1254. 15. Lomas-Lopez R, Paracuellos P, Riberty M, Cozzone AJ, Duclos B (2007) Several enzymes of the central metabolism are phosphorylated in Staphylococ- cus aureus. FEMS Microbiol Lett 272: 35–42. 16. Donat S, Streker K, Schirmeister T, Rakette S, Stehle T, et al. (2009) Transcriptome and functional analysis of the eukaryotic-type serine/threonine kinase PknB in Staphylococcus aureus. J Bacteriol 191: 4056–4069. 17. Debarbouille M, Dramsi S, Dussurget O, Nahori MA, Vaganay E, et al. (2009) Characterization of a serine/threonine kinase involved in virulence of Staphylococcus aureus. J Bacteriol 191: 4070–4081. 18. Deftereos SP, Michailidou E, Karagiannakis GK, Grigoriadi S, Prassopoulos P (2009) Hematogenous infantile infection presenting as osteomyelitis and septic arthritis: a case report. Cases J 2: 8293. 19. Geipel U (2009) Pathogenic organisms in hip joint infections. Int J Med Sci 6: 234–240. PknB Phosphorylation Profile Table S2 Peptides phosphorylated by PknB grouped according to function Found at: doi:10.1371/journal.pone.0009057.s002 (0.16 MB DOC) Acknowledgments The authors wish to thank Michel De´babouille´ and Tarek Msadek for useful discussions. Author Contributions Conceived and designed the experiments: MM AD DB MPP JMvD KO. Performed the experiments: MM SD SR TRHMK SHD AD ER KG. Analyzed the data: MM SD SHD AD DB MPP KO. Contributed reagents/materials/analysis tools: SR TS MPP KO. Wrote the paper: MM SD SR TS TRHMK SHD AD ER KG DB MPP JMvD KO. 20. Carrillo-Marquez MA, Hulten KG, Hammerman W, Mason EO, Kaplan SL (2009) USA300 is the predominant genotype causing Staphylococcus aureus septic arthritis in children. Pediatr Infect Dis J 28: 1076–1080. 21. Melly MA, Thomison JB, Rogers DE (1960) Fate of staphylococci within human leukocytes. J Exp Med 112: 1121–1130. 22. Kapral FA (1965) The phagocytosis and intracellular fate of staphylococci. Ann N Y Acad Sci 128: 285–300. 23. Rogers DE, Tompsett R (1952) The survival of staphylococci within human leukocytes. J Exp Med 95: 209–230. 24. Gresham HD, Lowrance JH, Caver TE, Wilson BS, Cheung AL, et al. (2000) Survival of Staphylococcus aureus inside neutrophils contributes to infection. J Immunol 164: 3713–3722. 25. Voyich JM, Braughton KR, Sturdevant DE, Whitney AR, Said-Salim B, et al. (2005) Insights into mechanisms used by Staphylococcus aureus to avoid destruction by human neutrophils. J Immunol 175: 3907–3919. 26. Baughn RE, Bonventre (1975) Nonspecific resistance to Listeria monocytogenes in mice infected and elicited with Staphylococcus aureus. Med Microbiol Immunol 161: 243–252. 27. Hebert A, Sayasith K, Senechal S, Dubreuil P, Lagace J (2000) Demonstration of intracellular Staphylococcus aureus in bovine mastitis alveolar cells and macrophages isolated from naturally infected cow milk. FEMS Microbiol Lett 193: 57–62. 28. Jakab GJ, Green GM (1976) Defect in intracellular killing of Staphylococcus aureus within alveolar macrophages in Sendai virus-infected murine lungs. J Clin Invest 57: 1533–1539. 29. Michailova L, Stoitsova S, Markova N, Kussovski V, Jordanova M, et al. (2000) Interaction of alveolar macrophages with Staphylococcus aureus and induction of microbial L-forms during infection in rats. Int J Med Microbiol 290: 259–267. 30. Elliott GR, Peterson PK, Verbrugh HA, Freiberg MR, Hoidal JR, et al. (1982) Influence of subinhibitory concentrations of penicillin, cephalothin, and clindamycin on Staphylococcus aureus growth in human phagocytic cells. Antimicrob Agents Chemother 22: 781–784. 31. Kubica M, Guzik K, Koziel J, Zarebski M, Richter W, et al. (2008) A potential new pathway for Staphylococcus aureus dissemination: the silent survival of S. aureus phagocytosed by human monocyte-derived macrophages. PLoS ONE 3: e1409. 32. Foster TJ, Hook M (1998) Surface protein adhesins of Staphylococcus aureus. Trends Microbiol 6: 484–488. 33. Lowy FD (1998) Staphylococcus aureus infections. N Engl J Med 339: 520–532. 34. Foster TJ (2005) Immune evasion by staphylococci. Nat Rev Microbiol 3: 948–958. 35. Sibbald MJ, Ziebandt AK, Engelmann S, Hecker M, de Jong A, et al. (2006) Mapping the pathways to staphylococcal pathogenesis by comparative secretomics. Microbiol Mol Biol Rev 70: 755–788. 36. Koziel J, Maciag-Gudowska A, Mikolajczyk T, Bzowska M, Sturdevant DE, et al. (2009) Phagocytosis of Staphylococcus aureus by macrophages exerts cytoprotective effects manifested by the upregulation of antiapoptotic factors. PLoS ONE 4: e5210. 37. Arbibe L, Mira JP, Teusch N, Kline L, Guha M, et al. (2000) Toll-like receptor 2-mediated NF-kappa B activation requires a Rac1-dependent pathway. Nat Immunol 1: 533–540. 38. Treusch AH, Vergin KL, Finlay LA, Donatz MG, Burton RM, et al. (2009) Seasonality and vertical structure of microbial communities in an ocean gyre. ISME J. 39. Walburger A, Koul A, Ferrari G, Nguyen L, Prescianotto-Baschong C, et al. (2004) Protein kinase G from pathogenic mycobacteria promotes survival within macrophages. Science 304: 1800–1804. PLoS ONE | www.plosone.org 7 February 2010 | Volume 5 | Issue 2 | e9057