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Abstract

EIIA is a member of a truncated phosphotransferase (PTS) system that serves regulatory functions and exists in many in addition to the sugar transport PTS. In , EIIA regulates K homeostasis through interaction with the K transporter TrkA and sensor kinase KdpD. In the H16, EIIA influences formation of the industrially important bioplastic poly(3-hydroxybutyrate) (PHB). PHB accumulation is controlled by the stringent response and induced under conditions of nitrogen deprivation. Knockout of EIIA increases the PHB content. In contrast, absence of enzyme I or HPr, which deliver phosphoryl groups to EIIA, has the opposite effect. To clarify the role of EIIA in PHB formation, we screened for interacting proteins that co-purify with Strep-tagged EIIA from cells. This approach identified the bifunctional ppGpp synthase/hydrolase SpoT1, a key enzyme of the stringent response. Two-hybrid and far-Western analyses confirmed the interaction and indicated that only non-phosphorylated EIIA interacts with SpoT1. Interestingly, this interaction does not occur between the corresponding proteins of . Vice versa, interaction of EIIA with KdpD appears to be absent in , although EIIA can perfectly substitute its homologue in in regulation of KdpD activity. Thus, interaction with KdpD might be an evolutionary ‘ancient’ task of EIIA that was subsequently replaced by interaction with SpoT1 in . In conclusion, EIIA might integrate information about nutritional status, as reflected by its phosphorylation state, into the stringent response, thereby controlling cellular PHB content in .

Funding
This study was supported by the:
  • DFG (Award GO1355/7-1)
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2014-04-01
2021-10-26
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References

  1. Bahr T., Lüttmann D., März W., Rak B., Görke B. ( 2011). Insight into bacterial phosphotransferase system-mediated signaling by interspecies transplantation of a transcriptional regulator. J Bacteriol 193:2013–2026 [View Article][PubMed]
    [Google Scholar]
  2. Bi W., Stambrook P. J. ( 1998). Site-directed mutagenesis by combined chain reaction. Anal Biochem 256:137–140 [View Article][PubMed]
    [Google Scholar]
  3. Blum H., Beier H., Gross H. J. ( 1987). Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8:93–99 [View Article]
    [Google Scholar]
  4. Boutte C. C., Crosson S. ( 2013). Bacterial lifestyle shapes stringent response activation. Trends Microbiol 21:174–180 [View Article][PubMed]
    [Google Scholar]
  5. Brigham C. J., Speth D. R., Rha C., Sinskey A. J. ( 2012). Whole-genome microarray and gene deletion studies reveal regulation of the polyhydroxyalkanoate production cycle by the stringent response in Ralstonia eutropha H16. Appl Environ Microbiol 78:8033–8044 [View Article][PubMed]
    [Google Scholar]
  6. Chavarría M., Kleijn R. J., Sauer U., Pflüger-Grau K., de Lorenzo V. ( 2012). Regulatory tasks of the phosphoenolpyruvate-phosphotransferase system of Pseudomonas putida in central carbon metabolism. MBio 3:e00028-12 [View Article][PubMed]
    [Google Scholar]
  7. Deutscher J. ( 2008). The mechanisms of carbon catabolite repression in bacteria. Curr Opin Microbiol 11:87–93 [View Article][PubMed]
    [Google Scholar]
  8. Deutscher J., Francke C., Postma P. W. ( 2006). How phosphotransferase system-related protein phosphorylation regulates carbohydrate metabolism in bacteria. Microbiol Mol Biol Rev 70:939–1031 [View Article][PubMed]
    [Google Scholar]
  9. Dissmeyer N., Schnittger A. ( 2011). Use of phospho-site substitutions to analyze the biological relevance of phosphorylation events in regulatory networks. Methods Mol Biol 779:93–138 [View Article][PubMed]
    [Google Scholar]
  10. Doucette C. D., Schwab D. J., Wingreen N. S., Rabinowitz J. D. ( 2011). α-Ketoglutarate coordinates carbon and nitrogen utilization via enzyme I inhibition. Nat Chem Biol 7:894–901 [View Article][PubMed]
    [Google Scholar]
  11. Dozot M., Poncet S., Nicolas C., Copin R., Bouraoui H., Mazé A., Deutscher J., De Bolle X., Letesson J. J. ( 2010). Functional characterization of the incomplete phosphotransferase system (PTS) of the intracellular pathogen Brucella melitensis.. PLoS ONE 5:e12679 [View Article][PubMed]
    [Google Scholar]
  12. Göpel Y., Papenfort K., Reichenbach B., Vogel J., Görke B. ( 2013). Targeted decay of a regulatory small RNA by an adaptor protein for RNase E and counteraction by an anti-adaptor RNA. Genes Dev 27:552–564 [View Article][PubMed]
    [Google Scholar]
  13. Görke B., Stülke J. ( 2008). Carbon catabolite repression in bacteria: many ways to make the most out of nutrients. Nat Rev Microbiol 6:613–624 [View Article][PubMed]
    [Google Scholar]
  14. Herzberg C., Weidinger L. A., Dörrbecker B., Hübner S., Stülke J., Commichau F. M. ( 2007). SPINE: a method for the rapid detection and analysis of protein-protein interactions in vivo.. Proteomics 7:4032–4035 [View Article][PubMed]
    [Google Scholar]
  15. Jahn S., Haverkorn van Rijsewijk B. R., Sauer U., Bettenbrock K. ( 2013). A role for EIIANtr in controlling fluxes in the central metabolism of E. coli K12. Biochim Biophys Acta 1833:2879–2889 [View Article][PubMed]
    [Google Scholar]
  16. Jault J. M., Fieulaine S., Nessler S., Gonzalo P., Di Pietro A., Deutscher J., Galinier A. ( 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 [View Article][PubMed]
    [Google Scholar]
  17. Jeffke T., Gropp N. H., Kaiser C., Grzeszik C., Kusian B., Bowien B. ( 1999). Mutational analysis of the cbb operon (CO2 assimilation) promoter of Ralstonia eutropha.. J Bacteriol 181:4374–4380[PubMed]
    [Google Scholar]
  18. Joyet P., Bouraoui H., Aké F. M., Derkaoui M., Zébré A. C., Cao T. N., Ventroux M., Nessler S., Noirot-Gros M. F. & other authors ( 2013). Transcription regulators controlled by interaction with enzyme IIB components of the phosphoenolpyruvate: sugar phosphotransferase system. Biochim Biophys Acta 1834:1415–1424 [View Article][PubMed]
    [Google Scholar]
  19. Kaddor C., Steinbüchel A. ( 2011). Effects of homologous phosphoenolpyruvate-carbohydrate phosphotransferase system proteins on carbohydrate uptake and poly(3-hydroxybutyrate) accumulation in Ralstonia eutropha H16. Appl Environ Microbiol 77:3582–3590 [View Article][PubMed]
    [Google Scholar]
  20. Kaddor C., Voigt B., Hecker M., Steinbüchel A. ( 2012). Impact of the core components of the phosphoenolpyruvate-carbohydrate phosphotransferase system, HPr and EI, on differential protein expression in Ralstonia eutropha H16. J Proteome Res 11:3624–3636 [View Article][PubMed]
    [Google Scholar]
  21. Karimova G., Pidoux J., Ullmann A., Ladant D. ( 1998). A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A 95:5752–5756 [View Article][PubMed]
    [Google Scholar]
  22. Kim M. S., Lee H., Heo L., Lim A., Seok C., Shin D. H. ( 2013). New molecular interaction of IIANtr and HPr from Burkholderia pseudomallei identified by X-ray crystallography and docking studies. Proteins 81:1499–1508 [View Article][PubMed]
    [Google Scholar]
  23. Kovach M. E., Elzer P. H., Hill D. S., Robertson G. T., Farris M. A., Roop R. M. II, Peterson K. M. ( 1995). Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176 [View Article][PubMed]
    [Google Scholar]
  24. Krauße D., Hunold K., Kusian B., Lenz O., Stülke J., Bowien B., Deutscher J. ( 2009). Essential role of the hprK gene in Ralstonia eutropha H16. J Mol Microbiol Biotechnol 17:146–152 [View Article][PubMed]
    [Google Scholar]
  25. Landmann J. J., Busse R. A., Latz J. H., Singh K. D., Stülke J., Görke B. ( 2011). Crh, the paralogue of the phosphocarrier protein HPr, controls the methylglyoxal bypass of glycolysis in Bacillus subtilis.. Mol Microbiol 82:770–787 [View Article][PubMed]
    [Google Scholar]
  26. Lee C. R., Cho S. H., Yoon M. J., Peterkofsky A., Seok Y. J. ( 2007). Escherichia coli enzyme IIANtr regulates the K+ transporter TrkA. Proc Natl Acad Sci U S A 104:4124–4129 [View Article][PubMed]
    [Google Scholar]
  27. Lee C. R., Cho S. H., Kim H. J., Kim M., Peterkofsky A., Seok Y. J. ( 2010). Potassium mediates Escherichia coli enzyme IIANtr-dependent regulation of sigma factor selectivity. Mol Microbiol 78:1468–1483 [View Article][PubMed]
    [Google Scholar]
  28. Lee C. R., Park Y. H., Kim M., Kim Y. R., Park S., Peterkofsky A., Seok Y. J. ( 2013). Reciprocal regulation of the autophosphorylation of enzyme INtr by glutamine and α-ketoglutarate in Escherichia coli. Mol Microbiol 88:473–485 [View Article][PubMed]
    [Google Scholar]
  29. Lüttmann D., Heermann R., Zimmer B., Hillmann A., Rampp I. S., Jung K., Görke B. ( 2009). Stimulation of the potassium sensor KdpD kinase activity by interaction with the phosphotransferase protein IIANtr in Escherichia coli.. Mol Microbiol 72:978–994 [View Article][PubMed]
    [Google Scholar]
  30. Lüttmann D., Göpel Y., Görke B. ( 2012). The phosphotransferase protein EIIANtr modulates the phosphate starvation response through interaction with histidine kinase PhoR in Escherichia coli.. Mol Microbiol 86:96–110 [View Article][PubMed]
    [Google Scholar]
  31. Miller J. ( 1972). Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  32. Neumann S., Grosse K., Sourjik V. ( 2012). Chemotactic signaling via carbohydrate phosphotransferase systems in Escherichia coli.. Proc Natl Acad Sci U S A 109:12159–12164 [View Article][PubMed]
    [Google Scholar]
  33. Pflüger K., de Lorenzo V. ( 2007). Growth-dependent phosphorylation of the PtsN (EIINtr) protein of Pseudomonas putida.. J Biol Chem 282:18206–18211 [View Article][PubMed]
    [Google Scholar]
  34. Pflüger-Grau K., Görke B. ( 2010). Regulatory roles of the bacterial nitrogen-related phosphotransferase system. Trends Microbiol 18:205–214 [View Article][PubMed]
    [Google Scholar]
  35. Pflüger-Grau K., Chavarría M., de Lorenzo V. ( 2011). The interplay of the EIIANtr component of the nitrogen-related phosphotransferase system (PTSNtr) of Pseudomonas putida with pyruvate dehydrogenase. Biochim Biophys Acta 1810:995–1005 [View Article][PubMed]
    [Google Scholar]
  36. Pohlmann A., Fricke W. F., Reinecke F., Kusian B., Liesegang H., Cramm R., Eitinger T., Ewering C., Pötter M. & other authors ( 2006). Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16. Nat Biotechnol 24:1257–1262 [View Article][PubMed]
    [Google Scholar]
  37. Potrykus K., Cashel M. ( 2008). (p)ppGpp: still magical?. Annu Rev Microbiol 62:35–51 [View Article][PubMed]
    [Google Scholar]
  38. Powell B. S., Court D. L., Inada T., Nakamura Y., Michotey V., Cui X., Reizer A., Saier M. H. Jr, Reizer J. ( 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 [View Article][PubMed]
    [Google Scholar]
  39. Prell J., Mulley G., Haufe F., White J. P., Williams A., Karunakaran R., Downie J. A., Poole P. S. ( 2012). The PTSNtr system globally regulates ATP-dependent transporters in Rhizobium leguminosarum.. Mol Microbiol 84:117–129 [View Article][PubMed]
    [Google Scholar]
  40. Pries A., Priefert H., Krüger N., Steinbüchel A. ( 1991). Identification and characterization of two Alcaligenes eutrophus gene loci relevant to the poly(β-hydroxybutyric acid)-leaky phenotype which exhibit homology to ptsH and ptsI of Escherichia coli.. J Bacteriol 173:5843–5853[PubMed]
    [Google Scholar]
  41. Rabus R., Reizer J., Paulsen I., Saier M. H. Jr ( 1999). Enzyme INtr 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 [View Article][PubMed]
    [Google Scholar]
  42. Reinecke F., Steinbüchel A. ( 2009). Ralstonia eutropha strain H16 as model organism for PHA metabolism and for biotechnological production of technically interesting biopolymers. J Mol Microbiol Biotechnol 16:91–108 [View Article][PubMed]
    [Google Scholar]
  43. Ruiz J. A., López N. I., Fernández R. O., Méndez B. S. ( 2001). Polyhydroxyalkanoate degradation is associated with nucleotide accumulation and enhances stress resistance and survival of Pseudomonas oleovorans in natural water microcosms. Appl Environ Microbiol 67:225–230 [View Article][PubMed]
    [Google Scholar]
  44. Segura D., Espín G. ( 1998). Mutational inactivation of a gene homologous to Escherichia coli ptsP affects poly-β-hydroxybutyrate accumulation and nitrogen fixation in Azotobacter vinelandii.. J Bacteriol 180:4790–4798[PubMed]
    [Google Scholar]
  45. Simon R., Priefer U., Pühler A. ( 1983). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Nat Biotechnol 1:784–791 [View Article]
    [Google Scholar]
  46. Singh K. D., Schmalisch M. H., Stülke J., Görke B. ( 2008). Carbon catabolite repression in Bacillus subtilis: quantitative analysis of repression exerted by different carbon sources. J Bacteriol 190:7275–7284 [View Article][PubMed]
    [Google Scholar]
  47. Srivastava S., Urban M., Friedrich B. ( 1982). Mutagenesis of Alcaligenes eutrophus by insertion of the drug-resistance transposon Tn5.. Arch Microbiol 131:203–207 [View Article][PubMed]
    [Google Scholar]
  48. Velázquez F., Pflüger K., Cases I., De Eugenio L. I., de Lorenzo V. ( 2007). The phosphotransferase system formed by PtsP, PtsO, and PtsN proteins controls production of polyhydroxyalkanoates in Pseudomonas putida.. J Bacteriol 189:4529–4533 [View Article][PubMed]
    [Google Scholar]
  49. Windhövel U., Bowien B. ( 1990). On the operon structure of the cfx gene clusters in Alcaligenes eutrophus.. Arch Microbiol 154:85–91 [View Article][PubMed]
    [Google Scholar]
  50. Ymele-Leki P., Houot L., Watnick P. I. ( 2013). Mannitol and the mannitol-specific enzyme IIB subunit activate Vibrio cholerae biofilm formation. Appl Environ Microbiol 79:4675–4683 [View Article][PubMed]
    [Google Scholar]
  51. Zimmer B., Hillmann A., Görke B. ( 2008). Requirements for the phosphorylation of the Escherichia coli EIIANtr protein in vivo.. FEMS Microbiol Lett 286:96–102 [View Article][PubMed]
    [Google Scholar]
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