1887

Abstract

In Gram-negative bacteria, tyrosine phosphorylation has been shown to play a role in the control of exopolysaccharide (EPS) production. This study demonstrated that the chromosomal ORF SMc02309 from 2011 encodes a protein with significant sequence similarity to low molecular mass protein-tyrosine phosphatases (LMW-PTPs), such as the Wzb. Unlike other well-characterized EPS biosynthesis gene clusters, which contain neighbouring LMW-PTPs and kinase, the succinoglycan (EPS I) gene cluster located on megaplasmid pSymB does not encode a phosphatase. Biochemical assays revealed that the SMc02309 protein hydrolyses -nitrophenyl phosphate (-NPP) with kinetic parameters similar to other bacterial LMW-PTPs. Furthermore, we show evidence that SMc02309 is not the LMW-PTP of the bacterial tyrosine-kinase (BY-kinase) ExoP. Nevertheless, ExoN, a UDP-glucose pyrophosphorylase involved in the first stages of EPS I biosynthesis, is phosphorylated at tyrosine residues and constitutes an endogenous substrate of the SMc02309 protein. Additionally, we show that the UDP-glucose pyrophosphorylase activity is modulated by SMc02309-mediated tyrosine dephosphorylation. Moreover, a mutation in the SMc02309 gene decreases EPS I production and delays nodulation on roots.

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2016-03-01
2021-10-22
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References

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. 1990; Basic local alignment search tool. J Mol Biol 215:403–410 [View Article][PubMed]
    [Google Scholar]
  2. Anderson C. R., Cook G. M. 2004; Isolation and characterization of arsenate-reducing bacteria from arsenic-contaminated sites in New Zealand. Curr Microbiol 48:341–347 [View Article][PubMed]
    [Google Scholar]
  3. Arakawa Y., Wacharotayankun R., Nagatsuka T., Ito H., Kato N., Ohta M. 1995; Genomic organization of the Klebsiella pneumoniae cps region responsible for serotype K2 capsular polysaccharide synthesis in the virulent strain Chedid. J Bacteriol 177:1788–1796[PubMed]
    [Google Scholar]
  4. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. (editors) 1995 Current Protocols in Molecular Biology New York: Wiley;
    [Google Scholar]
  5. Battisti L., Lara J. C., Leigh J. A. 1992; Specific oligosaccharide form of the Rhizobium meliloti exopolysaccharide promotes nodule invasion in alfalfa. Proc Natl Acad Sci U S A 89:5625–5629 [View Article][PubMed]
    [Google Scholar]
  6. Becker A., Kleickmann A., Keller M., Arnold W., Pühler A. 1993; Identification and analysis of the Rhizobium meliloti exoAMONP genes involved in exopolysaccharide biosynthesis and mapping of promoters located on the exoHKLAMONP fragment. Mol Gen Genet 241:367–379[PubMed]
    [Google Scholar]
  7. Becker A., Niehaus K., Pühler A. 1995; Low-molecular-weight succinoglycan is predominantly produced by Rhizobium meliloti strains carrying a mutated ExoP protein characterized by a periplasmic N-terminal domain and a missing C-terminal domain. Mol Microbiol 16:191–204 [View Article][PubMed]
    [Google Scholar]
  8. Bennett M. S., Guan Z., Laurberg M., Su X. D. 2001; Bacillus subtilis arsenate reductase is structurally and functionally similar to low molecular weight protein tyrosine phosphatases. Proc Natl Acad Sci U S A 98:13577–13582 [View Article][PubMed]
    [Google Scholar]
  9. Bergmeyer H. U., Graßl M., Walter H.-E. 1983; Uridinediphosphoglucose pyrophosphorylase. In Methods in Enzymatic Analysis, 3rd edn.. vol. 2 pp 324–326 Edited by Bergmeyer H. U. Weinheim: Verlag Chemie;
    [Google Scholar]
  10. Beringer J. E. 1981; R factor transfer in Rhizobium leguminosarum . J Gen Microbiol 84:188–198[PubMed]
    [Google Scholar]
  11. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 [View Article][PubMed]
    [Google Scholar]
  12. Bugert P., Geider K. 1995; Molecular analysis of the ams operon required for exopolysaccharide synthesis of Erwinia amylovora . Mol Microbiol 15:917–933 [View Article][PubMed]
    [Google Scholar]
  13. Bugert P., Geider K. 1997; Characterization of the amsI gene product as a low molecular weight acid phosphatase controlling exopolysaccharide synthesis of Erwinia amylovora . FEBS Lett 400:252–256 [View Article][PubMed]
    [Google Scholar]
  14. Cirri P., Chiarugi P., Camici G., Manao G., Raugei G., Cappugi G., Ramponi G. 1993; The role of Cys12, Cys17 and Arg18 in the catalytic mechanism of low-Mr cytosolic phosphotyrosine protein phosphatase. Eur J Biochem 214:647–657 [View Article][PubMed]
    [Google Scholar]
  15. Cozzone A. J. 2005; Role of protein phosphorylation on serine/threonine and tyrosine in the virulence of bacterial pathogens. J Mol Microbiol Biotechnol 9:198–213 [View Article][PubMed]
    [Google Scholar]
  16. Daniels R., Reynaert S., Hoekstra H., Verreth C., Janssens J., Braeken K., Fauvart M., Beullens S., Heusdens C., other authors. 2006; Quorum signal molecules as biosurfactants affecting swarming in Rhizobium etli . Proc Natl Acad Sci U S A 103:14965–14970 [View Article][PubMed]
    [Google Scholar]
  17. Dische Z. 1962; General color reactions. Methods Carbohydr Chem 1:478–492
    [Google Scholar]
  18. Eramian D., Eswar N., Shen M. Y., Sali A. 2008; How well can the accuracy of comparative protein structure models be predicted?. Protein Sci 17:1881–1893 [View Article][PubMed]
    [Google Scholar]
  19. Ferreira A. S., Leitão J. H., Sousa S. A., Cosme A. M., Sá-Correia I., Moreira L. M. 2007; Functional analysis of Burkholderia cepacia genes bceD and bceF, encoding a phosphotyrosine phosphatase and a tyrosine autokinase, respectively: role in exopolysaccharide biosynthesis and biofilm formation. Appl Environ Microbiol 73:524–534 [View Article][PubMed]
    [Google Scholar]
  20. Geddes B. A., González J. E., Oresnik I. J. 2014; Exopolysaccharide production in response to medium acidification is correlated with an increase in competition for nodule occupancy. Mol Plant Microbe Interact 27:1307–1317 [View Article][PubMed]
    [Google Scholar]
  21. Glucksmann M. A., Reuber T. L., Walker G. C. 1993; Genes needed for the modification, polymerization, export, and processing of succinoglycan by Rhizobium meliloti: a model for succinoglycan biosynthesis. J Bacteriol 175:7045–7055[PubMed]
    [Google Scholar]
  22. González J. E., Reuhs B. L., Walker G. C. 1996; Low molecular weight EPS II of Rhizobium meliloti allows nodule invasion in Medicago sativa . Proc Natl Acad Sci U S A 93:8636–8641 [View Article][PubMed]
    [Google Scholar]
  23. Grangeasse C., Doublet P., Vincent C., Vaganay E., Riberty M., Duclos B., Cozzone A. J. 1998; Functional characterization of the low-molecular-mass phosphotyrosine-protein phosphatase of Acinetobacter johnsonii . J Mol Biol 278:339–347 [View Article][PubMed]
    [Google Scholar]
  24. Grangeasse C., Obadia B., Mijakovic I., Deutscher J., Cozzone A. J., Doublet P. 2003; Autophosphorylation of the Escherichia coli protein kinase Wzc regulates tyrosine phosphorylation of Ugd, a UDP-glucose dehydrogenase. J Biol Chem 278:39323–39329 [View Article][PubMed]
    [Google Scholar]
  25. Grangeasse C., Cozzone A. J., Deutscher J., Mijakovic I. 2007; Tyrosine phosphorylation: an emerging regulatory device of bacterial physiology. Trends Biochem Sci 32:86–94 [View Article][PubMed]
    [Google Scholar]
  26. Gruszczyk J., Fleurie A., Olivares-Illana V., Béchet E., Zanella-Cleon I., Moréra S., Meyer P., Pompidor G., Kahn R., other authors. 2011; Structure analysis of the Staphylococcus aureus UDP-N-acetyl-mannosamine dehydrogenase Cap5O involved in capsular polysaccharide biosynthesis. J Biol Chem 286:17112–17121 [View Article][PubMed]
    [Google Scholar]
  27. Huang J., Schell M. 1995; Molecular characterization of the eps gene cluster of Pseudomonas solanacearum and its transcriptional regulation at a single promoter. Mol Microbiol 16:977–989 [View Article][PubMed]
    [Google Scholar]
  28. Jofré E., Becker A. 2009; Production of succinoglycan polymer in Sinorhizobium meliloti is affected by SMb21506 and requires the N-terminal domain of ExoP. Mol Plant Microbe Interact 22:1656–1668 [View Article][PubMed]
    [Google Scholar]
  29. Jones K. M. 2012; Increased production of the exopolysaccharide succinoglycan enhances Sinorhizobium meliloti 1021 symbiosis with the host plant Medicago truncatula . J Bacteriol 194:4322–4331 [View Article][PubMed]
    [Google Scholar]
  30. Keller M., Roxlau A., Weng W. M., Schmidt M., Quandt J., Niehaus K., Jording D., Arnold W., Pühler A. 1995; Molecular analysis of the Rhizobium meliloti mucR gene regulating the biosynthesis of the exopolysaccharides succinoglycan and galactoglucan. Mol Plant Microbe Interact 8:267–277 [View Article][PubMed]
    [Google Scholar]
  31. Kennelly P. J. 2001; Protein phosphatases—a phylogenetic perspective. Chem Rev 101:2291–2312 [View Article][PubMed]
    [Google Scholar]
  32. Kennelly P. J. 2002; Protein kinases and protein phosphatases in prokaryotes: a genomic perspective. FEMS Microbiol Lett 206:1–8 [View Article][PubMed]
    [Google Scholar]
  33. Kennelly P. J., Potts M. 1999; Life among the primitives: protein O-phosphatases in prokaryotes. Front Biosci 4:d372–d385 [View Article][PubMed]
    [Google Scholar]
  34. Lacour S., Bechet E., Cozzone A. J., Mijakovic I., Grangeasse C. 2008; Tyrosine phosphorylation of the UDP-glucose dehydrogenase of Escherichia coli is at the crossroads of colanic acid synthesis and polymyxin resistance. PLoS One 3:e3053 [View Article][PubMed]
    [Google Scholar]
  35. Leigh J. A., Lee C. C. 1988; Characterization of polysaccharides of Rhizobium meliloti exo mutants that form ineffective nodules. J Bacteriol 170:3327–3332[PubMed]
    [Google Scholar]
  36. Leigh J. A., Walker G. C. 1994; Exopolysaccharides of Rhizobium: synthesis, regulation and symbiotic function. Trends Genet 10:63–67 [View Article][PubMed]
    [Google Scholar]
  37. Markowitz V. M., Korzeniewski F., Palaniappan K., Szeto E., Werner G., Padki A., Zhao X., Dubchak I., Hugenholtz P., other authors. 2006; The integrated microbial genomes (IMG) system. Nucleic Acids Res 34:D344–D348 [View Article][PubMed]
    [Google Scholar]
  38. Mijakovic I., Musumeci L., Tautz L., Petranovic D., Edwards R. A., Jensen P. R., Mustelin T., Deutscher J., Bottini N. 2005a; In vitro characterization of the Bacillus subtilis protein tyrosine phosphatase YwqE. J Bacteriol 187:3384–3390 [View Article][PubMed]
    [Google Scholar]
  39. Mijakovic I., Petranovic D., Bottini N., Deutscher J., Ruhdal Jensen P. 2005b; Protein-tyrosine phosphorylation in Bacillus subtilis . J Mol Microbiol Biotechnol 9:189–197 [View Article][PubMed]
    [Google Scholar]
  40. Minic Z., Marie C., Delorme C., Faurie J. M., Mercier G., Ehrlich D., Renault P. 2007; Control of EpsE, the phosphoglycosyltransferase initiating exopolysaccharide synthesis in Streptococcus thermophilus, by EpsD tyrosine kinase. J Bacteriol 189:1351–1357 [View Article][PubMed]
    [Google Scholar]
  41. Moreno E., Stackebrandt E., Dorsch M., Wolters J., Busch M., Mayer H. 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[PubMed]
    [Google Scholar]
  42. Mori Y., Maeda M., Takegawa K., Kimura Y. 2012; PhpA, a tyrosine phosphatase of Myxococcus xanthus, is involved in the production of exopolysaccharide. Microbiology 158:2546–2555 [View Article][PubMed]
    [Google Scholar]
  43. Müller P., Hynes M., Kapp D., Niehaus K., Pühler A. 1988; Two classes of Rhizobium meliloti infection mutants differ in exopolysaccharide production and in coinoculation properties with nodulation mutants. Mol Gen Genet 211:17–26 [View Article]
    [Google Scholar]
  44. Nadler C., Koby S., Peleg A., Johnson A. C., Suddala K. C., Sathiyamoorthy K., Smith B. E., Saper M. A., Rosenshine I. 2012; Cycling of Etk and Etp phosphorylation states is involved in formation of group 4 capsule by Escherichia coli . PLoS One 7:e37984 [View Article][PubMed]
    [Google Scholar]
  45. Nakar D., Gutnick D. L. 2003; Involvement of a protein tyrosine kinase in production of the polymeric bioemulsifier emulsan from the oil-degrading strain Acinetobacter lwoffii RAG-1. J Bacteriol 185:1001–1009 [View Article][PubMed]
    [Google Scholar]
  46. Niehaus K., Becker A. 1998; The role of microbial surface polysaccharides in the Rhizobium-legume interaction. Subcell Biochem 29:73–116 [View Article][PubMed]
    [Google Scholar]
  47. Niemeyer D., Becker A. 2001; The molecular weight distribution of succinoglycan produced by Sinorhizobium meliloti is influenced by specific tyrosine phosphorylation and ATPase activity of the cytoplasmic domain of the ExoP protein. J Bacteriol 183:5163–5170 [View Article][PubMed]
    [Google Scholar]
  48. Paiment A., Hocking J., Whitfield C. 2002; Impact of phosphorylation of specific residues in the tyrosine autokinase, Wzc, on its activity in assembly of group 1 capsules in Escherichia coli . J Bacteriol 184:6437–6447 [View Article][PubMed]
    [Google Scholar]
  49. Preneta R., Jarraud S., Vincent C., Doublet P., Duclos B., Etienne J., Cozzone A. J. 2002; Isolation and characterization of a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase from Klebsiella pneumoniae . Comp Biochem Physiol B Biochem Mol Biol 131:103–112 [View Article][PubMed]
    [Google Scholar]
  50. Reed J. W., Capage M., Walker G. C. 1991; Rhizobium meliloti exoG and exoJ mutations affect the exoX-exoY system for modulation of exopolysaccharide production. J Bacteriol 173:3776–3788[PubMed]
    [Google Scholar]
  51. Reuber T. L., Walker G. C. 1993; Biosynthesis of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti . Cell 74:269–280 [View Article][PubMed]
    [Google Scholar]
  52. Rolfe B. G., Gresshoff P. M., Shine J. 1980; Rapid screening for symbiotic mutants of Rhizobium and white clover. Plant Sci Lett 19:277–284 [View Article]
    [Google Scholar]
  53. Šali A., Potterton L., Yuan F., van Vlijmen H., Karplus M. 1995; Evaluation of comparative protein modeling by modeller . Proteins 23:318–326 [View Article][PubMed]
    [Google Scholar]
  54. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: A Laboratory Manual, 2nd edn Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press;
    [Google Scholar]
  55. Shi L., Potts M., Kennelly P. J. 1998; The serine, threonine, and/or tyrosine-specific protein kinases and protein phosphatases of prokaryotic organisms: a family portrait. FEMS Microbiol Rev 22:229–253 [View Article][PubMed]
    [Google Scholar]
  56. Skorupska A., Janczarek M., Marczak M., Mazur A., Król J. 2006; Rhizobial exopolysaccharides: genetic control and symbiotic functions. Microb Cell Fact 5:7–26 [View Article][PubMed]
    [Google Scholar]
  57. Söding J., Biegert A., Lupas A. N. 2005; The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res 33: Web Server W244–W248 [View Article][PubMed]
    [Google Scholar]
  58. Standish A. J., Morona R. 2014; The role of bacterial protein tyrosine phosphatases in the regulation of the biosynthesis of secreted polysaccharides. Antioxid Redox Signal 20:2274–2289 [View Article][PubMed]
    [Google Scholar]
  59. Standish A. J., Salim A. A., Zhang H., Capon R. J., Morona R. 2012; Chemical inhibition of bacterial protein tyrosine phosphatase suppresses capsule production. PLoS One 7:e36312 [View Article][PubMed]
    [Google Scholar]
  60. Tan H., Wan S., Liu P. Q., Wang L., Zhang C. C., Chen W. L. 2013; Alr5068, a low-molecular-weight protein tyrosine phosphatase, is involved in formation of the heterocysts polysaccharide layer in the cyanobacterium Anabaena sp. PCC 7120. Res Microbiol 164:875–885 [View Article][PubMed]
    [Google Scholar]
  61. Tauch A., Zheng Z., Pühler A., Kalinowski J. 1998; Corynebacterium striatum chloramphenicol resistance transposon Tn5564: genetic organization and transposition in Corynebacterium glutamicum . Plasmid 40:126–139 [View Article][PubMed]
    [Google Scholar]
  62. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354 [View Article][PubMed]
    [Google Scholar]
  63. Vincent C., Doublet P., Grangeasse C., Vaganay E., Cozzone A. J., Duclos B. 1999; Cells of Escherichia coli contain a protein-tyrosine kinase, Wzc, and a phosphotyrosine-protein phosphatase, Wzb. J Bacteriol 181:3472–3477[PubMed]
    [Google Scholar]
  64. Vincent C., Duclos B., Grangeasse C., Vaganay E., Riberty M., Cozzone A. J., Doublet P. 2000; Relationship between exopolysaccharide production and protein-tyrosine phosphorylation in Gram-negative bacteria. J Mol Biol 304:311–321 [View Article][PubMed]
    [Google Scholar]
  65. Whitfield C. 2006; Biosynthesis and assembly of capsular polysaccharides in Escherichia coli . Annu Rev Biochem 75:39–68 [View Article][PubMed]
    [Google Scholar]
  66. Whitmore S. E., Lamont R. J. 2012; Tyrosine phosphorylation and bacterial virulence. Int J Oral Sci 4:1–6 [View Article][PubMed]
    [Google Scholar]
  67. Yother J. 2011; Capsules of Streptococcus pneumoniae and other bacteria: paradigms for polysaccharide biosynthesis and regulation. Annu Rev Microbiol 65:563–581 [View Article][PubMed]
    [Google Scholar]
  68. Zevenhuizen L.P.T.M., van Neerven A. R. W. 1983; (1,2)-β-d-Glucan and acidic oligosaccharides produced by Rhizobium meliloti . Carbohydr Res 118:127–134 [View Article]
    [Google Scholar]
  69. Zhang Z.-Y. 2001; Protein tyrosine phosphatases: prospects for therapeutics. Curr Opin Chem Biol 5:416–423 [View Article][PubMed]
    [Google Scholar]
  70. Zhang Z. Y., Wang Y., Wu L., Fauman E. B., Stuckey J. A., Schubert H. L., Saper M. A., Dixon J. E. 1994; The Cys(X)5Arg catalytic motif in phosphoester hydrolysis. Biochemistry 33:15266–15270 [View Article][PubMed]
    [Google Scholar]
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