Acknowledgments We thank C. Baron for his kind gift of polyclonal anti-VirB5 and -VirB10 antisera. The work of J. Mignolet in preparing the ORFeome library pool is acknowledged. We are grateful to J-F Dierick for fruitful discussions. References 1. Deutscher J, Francke C, Postma PW (2006) How phosphotransferase system- related protein phosphorylation regulates carbohydrate metabolism in bacteria. Microbiol Mol Biol Rev 70: 939–1031. 2. Gorke B, Stu¨ lke J (2008) Carbon catabolite repression in bacteria: many ways to make the most out of nutrients. Nat Rev Microbiol 6: 613–624. 3. Bru¨ ckner R, Titgemeyer F (2002) Carbon catabolite repression in bacteria: choice of the carbon source and autoregulatory limitation of sugar utilization. FEMS Microbiol Lett 209: 141–148. 4. Powell BS, Court DL, Inada T, Nakamura Y, Michotey V, et al. (1995) Novel proteins of the phosphotransferase system encoded within the rpoN operon of Escherichia coli. Enzyme IIANtr affects growth on organic nitrogen and the conditional lethality of an erats mutant. J Biol Chem 270: 4822–4839. 5. Rabus R, Reizer J, Paulsen I, Saier MH, Jr. (1999) Enzyme I(Ntr) from Escherichia coli. A novel enzyme of the phosphoenolpyruvate-dependent phosphotransferase system exhibiting strict specificity for its phosphoryl acceptor, NPr. J Biol Chem 274: 26185–26191. 6. Reizer J, Reizer A, Merrick MJ, Plunkett G, 3rd, Rose DJ, et al. (1996) Novel phosphotransferase-encoding genes revealed by analysis of the Escherichia coli genome: a chimeric gene encoding an Enzyme I homologue that possesses a putative sensory transduction domain. Gene 181: 103–108. 7. Tchieu JH, Norris V, Edwards JS, Saier MH Jr. (2001) The complete phosphotranferase system in Escherichia coli. J Mol Microbiol Biotechnol 3: 329–346. 8. Pflu¨ ger-Grau K, Go¨rke B (2010) Regulatory roles of the bacterial nitrogen- related phosphotransferase system. Trends Microbiol 18: 205–214. 9. Cases I, Perez-Martin J, de Lorenzo V (1999) The IIANtr (PtsN) protein of Pseudomonas putida mediates the C source inhibition of the sigma54-dependent Pu promoter of the TOL plasmid. J Biol Chem 274: 15562–15568. 10. Jones DH, Franklin FC, Thomas CM (1994) Molecular analysis of the operon which encodes the RNA polymerase sigma factor sigma 54 of Escherichia coli. Microbiology 140 (Pt 5): 1035–1043. 11. Merrick MJ, Coppard JR (1989) Mutations in genes downstream of the rpoN gene (encoding sigma 54) of Klebsiella pneumoniae affect expression from sigma 54- dependent promoters. Mol Microbiol 3: 1765–1775. 12. Michiels J, Van Soom T, D’Hooghe I, Dombrecht B, Benhassine T, et al. (1998) The Rhizobium etli rpoN locus: DNA sequence analysis and phenotypical characterization of rpoN, ptsN, and ptsA mutants. J Bacteriol 180: 1729–1740. 13. Aravind L, Ponting CP (1997) The GAF domain: an evolutionary link between diverse phototransducing proteins. Trends Biochem Sci 22: 458–459. 14. Lee CR, Koo BM, Cho SH, Kim YJ, Yoon MJ, et al. (2005) Requirement of the dephospho-form of enzyme IIANtr for derepression of Escherichia coli K-12 ilvBN expression. Mol Microbiol 58: 334–344. 15. Lu¨ ttmann D, Heermann R, Zimmer B, Hillmann A, Rampp IS, et al. (2009) Stimulation of the potassium sensor KdpD kinase activity by interaction with the phosphotransferase protein IIA(Ntr) in Escherichia coli. Mol Microbiol 72: 978–994. 16. Moreno E, Stackebrandt E, Dorsch M, Wolters J, Busch M, et al. (1990) Brucella abortus 16S rRNA and lipid A reveal a phylogenetic relationship with members of the alpha-2 subdivision of the class Proteobacteria. J Bacteriol 172: 3569–3576. 17. Boschiroli ML, Foulongne V, O’Callaghan D (2001) Brucellosis: a worldwide zoonosis. Curr Opin Microbiol 4: 58–64. 18. Young EJ (1995) An overview of human brucellosis. Clin Infect Dis 21: 283–289; quiz 290. 19. Delrue RM, Lestrate P, Tibor A, Letesson JJ, De Bolle X (2004) Brucella pathogenesis, genes identified from random large-scale screens. FEMS Microbiol Lett 231: 1–12. 20. Wu Q, Pei J, Turse C, Ficht TA (2006) Mariner mutagenesis of Brucella melitensis reveals genes with previously uncharacterized roles in virulence and survival. BMC Microbiol 6: 102. 21. Ko¨hler S, Foulongne V, Ouahrani-Bettache S, Bourg G, Teyssier J, et al. (2002) The analysis of the intramacrophagic virulome of Brucella suis deciphers the environment encountered by the pathogen inside the macrophage host cell. Proc Natl Acad Sci U S A 99: 15711–15716. 22. DelVecchio VG, Kapatral V, Redkar RJ, Patra G, Mujer C, et al. (2002) The genome sequence of the facultative intracellular pathogen Brucella melitensis. Proc Natl Acad Sci U S A 99: 443–448. 23. Halling SM, Peterson-Burch BD, Bricker BJ, Zuerner RL, Qing Z, et al. (2005) Completion of the genome sequence of Brucella abortus and comparison to the highly similar genomes of Brucella melitensis and Brucella suis. J Bacteriol 187: 2715–2726. 24. Paulsen IT, Seshadri R, Nelson KE, Eisen JA, Heidelberg JF, et al. (2002) The Brucella suis genome reveals fundamental similarities between animal and plant pathogens and symbionts. Proc Natl Acad Sci U S A 99: 13148–13153. Brucella PTS Author Contributions Conceived and designed the experiments: JD XDB JJL. Performed the experiments: MD SP CN RC HB AM. Analyzed the data: MD CN JD XDB JJL. Wrote the paper: MD JD JJL. Helped draft the manuscript: SP XDB. 25. Barabote RD, Saier MH Jr. (2005) Comparative genomic analyses of the bacterial phosphotransferase system. Microbiol Mol Biol Rev 69: 608–634. 26. Boe¨l G, Mijakovic I, Maze A, Poncet S, Taha MK, et al. (2003) Transcription regulators potentially controlled by HPr kinase/phosphorylase in Gram-negative bacteria. J Mol Microbiol Biotechnol 5: 206–215. 27. Hu KY, Saier MH Jr. (2002) Phylogeny of phosphoryl transfer proteins of the phosphoenolpyruvate-dependent sugar-transporting phosphotransferase system. Res Microbiol 153: 405–415. 28. Deutscher J, Pevec B, Beyreuther K, Kiltz HH, Hengstenberg W (1986) Streptococcal phosphoenolpyruvate-sugar phosphotransferase system: amino acid sequence and site of ATP-dependent phosphorylation of HPr. Biochemistry 25: 6543–6551. 29. Deutscher J, Saier MH Jr. (1983) ATP-dependent protein kinase-catalyzed phosphorylation of a seryl residue in HPr, a phosphate carrier protein of the phosphotransferase system in Streptococcus pyogenes. Proc Natl Acad Sci U S A 80: 6790–6794. 30. Poncet S, Mijakovic I, Nessler S, Gueguen-Chaignon V, Chaptal V, et al. (2004) HPr kinase/phosphorylase, a Walker motif A-containing bifunctional sensor enzyme controlling catabolite repression in Gram-positive bacteria. Biochim Biophys Acta 1697: 123–135. 31. Deutscher J, Kuster E, Bergstedt U, Charrier V, Hillen W (1995) Protein kinase- dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in gram-positive bacteria. Mol Microbiol 15: 1049–1053. 32. Pinedo CA, Gage DJ (2009) HPrK regulates succinate-mediated catabolite repression in the gram-negative symbiont Sinorhizobium meliloti. J Bacteriol 191: 298–309. 33. Charles TC, Nester EW (1993) A chromosomally encoded two-component sensory transduction system is required for virulence of Agrobacterium tumefaciens. J Bacteriol 175: 6614–6625. 34. Cheng HP, Walker GC (1998) Succinoglycan production by Rhizobium meliloti is regulated through the ExoS-ChvI two-component regulatory system. J Bacteriol 180: 20–26. 35. Sola-Landa A, Pizarro-Cerda J, Grillo MJ, Moreno E, Moriyon I, et al. (1998) A two-component regulatory system playing a critical role in plant pathogens and endosymbionts is present in Brucella abortus and controls cell invasion and virulence. Mol Microbiol 29: 125–138. 36. Sganga MW, Aksamit RR, Cantoni GL, Bauer CE (1992) Mutational and nucleotide sequence analysis of S-adenosyl-L-homocysteine hydrolase from Rhodobacter capsulatus. Proc Natl Acad Sci U S A 89: 6328–6332. 37. Sun J, Daniel R, Wagner-Dobler I, Zeng AP (2004) Is autoinducer-2 a universal signal for interspecies communication: a comparative genomic and phylogenetic analysis of the synthesis and signal transduction pathways. BMC Evol Biol 4: 36. 38. Liu K, Yu J, Russell DG (2003) pckA-deficient Mycobacterium bovis BCG shows attenuated virulence in mice and in macrophages. Microbiology 149: 1829–1835. 39. Liu P, Wood D, Nester EW (2005) Phosphoenolpyruvate carboxykinase is an acid-induced, chromosomally encoded virulence factor in Agrobacterium tumefa- ciens. J Bacteriol 187: 6039–6045. 40. Osteras M, Driscoll BT, Finan TM (1995) Molecular and expression analysis of the Rhizobium meliloti phosphoenolpyruvate carboxykinase (pckA) gene. J Bacteriol 177: 1452–1460. 41. Fieulaine S, Morera S, Poncet S, Monedero V, Gueguen-Chaignon V, et al. (2001) X-ray structure of HPr kinase: a bacterial protein kinase with a P-loop nucleotide-binding domain. Embo J 20: 3917–3927. 42. Monedero V, Poncet S, Mijakovic I, Fieulaine S, Dossonnet V, et al. (2001) Mutations lowering the phosphatase activity of HPr kinase/phosphatase switch off carbon metabolism. Embo J 20: 3928–3937. 43. Pelletier M, Lortie LA, Frenette M, Vadeboncoeur C (1998) The phosphoe- nolpyruvate:mannose phosphotransferase system of Streptococcus salivarius. Func- tional and biochemical characterization of IIABL(Man) and IIABH(Man). Biochemistry 37: 1604–1612. 44. Jault JM, Fieulaine S, Nessler S, Gonzalo P, Di Pietro A, et al. (2000) The HPr kinase from Bacillus subtilis is a homo-oligomeric enzyme which exhibits strong positive cooperativity for nucleotide and fructose 1,6-bisphosphate binding. J Biol Chem 275: 1773–1780. 45. Mijakovic I, Poncet S, Galinier A, Monedero V, Fieulaine S, et al. (2002) Pyrophosphate-producing protein dephosphorylation by HPr kinase/phosphor- ylase: a relic of early life? Proc Natl Acad Sci U S A 99: 13442–13447. 46. Dozot M, Boigegrain RA, Delrue RM, Hallez R, Ouahrani-Bettache S, et al. (2006) The stringent response mediator Rsh is required for Brucella melitensis and Brucella suis virulence, and for expression of the type IV secretion system virB. Cell Microbiol 8: 1791–1802. PLoS ONE | www.plosone.org 15 September 2010 | Volume 5 | Issue 9 | e12679