Metabolic regulation of Escherichia coli and its glnG and zwf mutants under nitrogen limitation (original) (raw)

Abstract

Background: It is quite important to understand how the central metabolism is regulated under nitrogen (N)limitation as well as carbon (C)-limitation. In particular, the effect of C/N ratio on the metabolism is of practical interest for the heterologous protein production, PHB production, etc. Although the carbon and nitrogen metabolisms are interconnected and the overall mechanism is complicated, it is strongly desirable to clarify the effects of culture environment on the metabolism from the practical application point of view. Results: The effect of C/N ratio on the metabolism in Escherichia coli was investigated in the aerobic continuous culture at the dilution rate of 0.2 h -1 based on fermentation data, transcriptional RNA level, and enzyme activity data. The glucose concentration was kept at 10 g/l, while ammonium sulfate concentration was varied from 5.94 to 0.594 g/l. The resultant C/N ratios were 1.68 (100%), 2.81(60%), 4.21(40%), 8.42(20%), and 16.84(10%), where the percentage values in brackets indicate the ratio of N-concentration as compared to the case of 5.94 g/l of ammonium sulfate. The mRNA levels of crp and mlc decreased, which caused ptsG transcript expression to be upregulated as C/N ratio increased. As C/N ratio increased cra transcript expression decreased, which caused ptsH, pfkA, and pykF to be up-regulated. At high C/N ratio, transcriptional mRNA level of soxR/S increased, which may be due to the activated respiratory chain as indicated by up-regulations of such genes as cyoA, cydB, ndh as well as the increase in the specific CO 2 production rate. The rpoN transcript expression increased with the increase in C/N ratio, which led glnA, L, G and gltD transcript expression to change in similar fashion. The nac transcript expression showed similar trend as rpoN, while gdhA transcript expression changed in reverse direction. The transcriptional mRNA level of glnB, which codes for P II , glnD and glnK increased as C/N ratio increases. It was shown that GS-GOGAT pathway was activated for gdhA mutant under N-rich condition. In the case of glnL mutant, GOGAT enzyme activity was reduced as compared to the wild type under N-limitation. In the case of gltB, D mutants, GDH and GS enzymes were utilized under both N-rich and N-limited conditions. In this case, the transcriptional mRNA level of gdhA and corresponding GDH enzyme activity was higher under N-limitation as compared to Nrich condition.

Loading...

Loading Preview

Sorry, preview is currently unavailable. You can download the paper by clicking the button above.

References (58)

  1. Hua Q, Yang C, Baba T, Mori H, Shimizu K: Response of the central metabolism in Escherichia coli to phosphoglucose isomerase and glucose-6-phosphate dehydrogenase knockouts. J Bacteriol 2003, 185:7053-7067.
  2. Hua Q, Yang C, Oshima T, Mori H, Shimizu K: Analysis of gene expression in Escherichia coli in response to changes of growth-limiting nutrient in chemostat cultures. Appl Environ Microbiol 2004, 70:2354-2366.
  3. Nanchen A, Schicker A, Revelles O, Sauer U: Cyclic AMP -dependent catabolite repression is dominant control mechanism of metabolic fluxes under glucose limitation. J Bacteriol 2008, 190:2323-2330.
  4. Sauer U, Eikmanns BJ: The PEP-pyruvate-oxaloacetate node as the switch point for carbon flux distribution in bacteria. FEMS Microbiol Rev 2005, 29:765-794.
  5. Reitzer L: Nitrogen assimilation and global regulation in Escherichia coli. Annu Rev Microbiol 2003, 57:155-176.
  6. Commichau FM, Forchhammer K, Stülke J: Regulatory links between carbon and nitrogen metabolism. Curr Opin Microbiol 2006, 9:167-172.
  7. Perrenound A, Sauer U: Impact of global transcriptional regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on glucose catabolism in Escherichia coli. J Bacteriol 2005, 187:3171-3179.
  8. Gosset G, Zhang Z, Nayyar S, Cuevas WA, Saier MH Jr: Transcriptome analysis of Crp-dependent catabolite control of gene expression in Escherichia coli. J Bacteriol 2004, 186:3516-3524.
  9. Mao XJ, Huo YX, Buck M, Kolb A, Wang YP: Interplay between CRP-cAMP and PII -Ntr systems forms novel regulatory network between carbon metabolism and nitrogen assimilation in Escherichia coli. Nucleic Acids Res 2007, 35:1432-1440.
  10. Yan D: Protection of the glutamate pool concentration in enteric bacteria. Proc Natl Acad Sci USA 2007, 104:9475-9480.
  11. Ninfa AJ, Jiang P, Atkinson MR, Peliska JA: Integration of antagonistic signals in the regulation of nitrogen assimilation in Escherichia coli. Curr Top Cell Reg 2000, 36:31-75.
  12. Fischer E, Sauer U: Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS. Eur J Biochem 2003, 270:880-891.
  13. Zhang X, Jantama K, Moore JC, Shanmugam KT, Ingram LO: Production of L-alanine by metabolically engineered Escherichia coli. Appl Microbiol Biotechnol 2007, 77:355-366.
  14. Koebmann BJ, Westerhoff HV, Snoep JL, Nilsson D, Jensen PR: The glycolytic flux in Escherichia coli is controlled by the demand for ATP. J Bacteriol 2002, 184:3909-3916.
  15. Jiang P, Ninfa AJ: Escherichia coli PII signal transduction protein controlling nitrogen assimilation acts as a sensor of adenylate energy charge in vitro. Biochemistry 2007, 46:12979-12996.
  16. Ninfa AJ, Jiang P: PII signal transduction proteins: sensors of α- ketoglutarate that regulate nitrogen metabolism. Curr Opin Microbiol 2005, 8:168-173.
  17. Kabir MM, Shimizu K: Fermentation characteristics and protein expression patterns in a recombinant Escherichia coli mutant lacking phosphoglucose isomerase for poly(3-hydroxybutyrate) production. Appl Microbiol Biotechnol 2003, 62:244-255.
  18. Shang F, Wen S, Wang X, Tan T: Effect of nitrogen limitation on the ergosterol production by fed-batch culture of Saccharomyces cerevisiae. J Biotechnol 2006, 122:285-292.
  19. Reitzer L, Schneider BL: Metabolic context and possible physiological themes of σ 54 -dependent genes in Escherichia coli. Microbiol Mol Biol Rev 2001, 65:422-444.
  20. Martínez I, Zhu J, Lin H, Bennett GN, San KY: Replacing Escherichia coli NAD-dependent glyceraldehyde3-phosphate dehydrogenase (GAPDH) with a NADP -dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways. Metab Eng 2008, 10:352-359.
  21. Yuan J, Doucette CD, Fowler WU, Feng X-J, Piazza M, Rabitz HA, Wingreen NS, Rabinowitz JD: Metabolomics-driven quantitative analysis of ammonia assimilation in E. coli. Mol Sys Biol 2009, 5:302.
  22. Rahman M, Hassan MR, Shimizu K: Growth phase-dependent changes in the expression of global regulatory genes and associated metabolic pathways in Escherichia coli. Biotechnol Lett 2008, 30:853-860.
  23. Zhang Z, Gosset G, Barabote R, Gonzalez CS, Cuevas WA, Saier MH Jr: Functional interactions between the carbon and iron utilization regulators, Crp and Fur, in Escherichia coli. J Bacteriol 2005, 187:980-990.
  24. Deutscher J, Francke C, Postma PW: How Phosphotransferase System- Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria. Microbiol Mol Biol Rev 2006, 70:939-1031.
  25. Helling RB: Pathway choice in glutamate synthesis in Escherichia coli. J Bacteriol 1998, 180:4571-4575.
  26. Liang A, Houghton RL: Coregulation of oxidized nicotinamide adenine dinucleotide (phosphate) transhydrogenase and glutamate dehydrogenase activities in enteric bacteria during nitrogen limitation. J Bacteriol 1981, 146:997-1002.
  27. Willis RC, Iwata KK, Furlong CE: Regulation of glutamine transport in Escherichia coli. J Bacteriol 1975, 122:1032-1037.
  28. Claverie-Martin F, Magasanik B: Role of integration host factor in the regulation of the glnHp2 promoter of Escherichia coli. Proc Nadl Acad Sci USA 1991, 88:1631-1635.
  29. Blauwkamp TA, Ninfa AJ: Physiological role of the GlnK signal transduction protein of Escherichia coli: survival of nitrogen starvation. Mol Microbiol 2002, 46:203-214.
  30. Magasanik B: Regulation of nitrogen utilization. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology Washington, DC: American Society for MicrobiologyNeidhardt FC, others 1996, 1344-1356.
  31. Riba L, Becerril B, Servèn-Gonzaèlez L, Valle F, Bolivar F: Identification of a functional promoter for the Escherichia coli gdhA gene and its regulation. Gene 1988, 71:233-246.
  32. Camarena L, Poggio S, Garcèa N, Osorio A: Transcriptional repression of gdhA in Escherichia coli is mediated by the Nac protein. FEMS Microbiol Lett 1998, 167:51-56.
  33. Ninfa AJ, Atkinson MR: PII signal transduction proteins. Trends Microbiol 2000, 8:172-179.
  34. Reitzer LJ, Magasanik B: Expression of glnA in Escherichia coli is regulated at tandem promoters. Proc Natl Acad Sci USA 1985, 82:1979-1983.
  35. Atkinson MR, Ninfa AJ: Mutational analysis of the bacterial signal transducing protein kinase/phosphatase nitrogen regulator II (NRII or NtrB). J Bacteriol 1993, 175:7016-7023.
  36. Atkinson MR, Blauwkamp TA, Ninfa AJ: Context-dependent functions of the PII and GlnK signal transduction proteins in Escherichia coli. J Bacteriol 2002, 184:5364-5375.
  37. van Heeswijk WC, Hoving S, Molenaar D, Stegeman B, Kahn D, Westerhoff HV: An alternative PII protein in the regulation of glutamine synthetase in Escherichia coli. Mol Microbiol 1996, 21:133-146.
  38. Pahel G, Rothstein DM, Magasanik B: Complex glnA-glnL-glnG operon of Escherichia coli. J Bacteriol 1982, 150:202-213.
  39. Paul L, Mishra PK, Blumenthal RM, Matthews RG: Integration of regulatory signals through involvement of multiple global regulators: control of the Escherichia coli gltBDF operon by Lrp, IHF, Crp, and ArgR. BMC Microbiol 2007, 7:2.
  40. Shapiro BM, Stadtman ER: Glutamine synthetase deadenylylating enzyme. Biochem Biophys Res Commun 1968, 30:32-37.
  41. Stadtman ER: Discovery of glutamine synthetase cascade. Methods Enzymol 1990, 182:793-809.
  42. Jaggi R, van Heeswijk WC, Westerhoff HV, Ollis DL, Vasudevan SG: The two opposing activities of adenylyltransferase reside in distinct homologous domains, with intramolecular signal transduction. EMBO J 1997, 16:5562-5571.
  43. Maheswaran M, Forchhammer K: Carbon-source-dependent nitrogen regulation in Escherichia coli is mediated through glutamine-dependent GlnB signalling. Microbiol 2003, 149:2163-2172.
  44. Merrick MJ, Edwards RA: Nitrogen control in bacteria. Microbiol Rev 1995, 4:604-622.
  45. Jiang P, Peliska JA, Ninfa AJ: The regulation of Escherichia coli glutamine synthetase revisited: role of 2-ketoglutarate in the regulation of glutamine synthetase adenylylation state. Biochemistry 1998, 37:12802-12810.
  46. Kustu S, Santero E, Keener J, Popham D, Weiss D: Expression of sigma 54 (ntrA)-dependent genes is probably united by a common mechanism. Microbiol Rev 1989, 53:367-376.
  47. Javelle A, Severi E, Thornton J, Merrick M: Ammonium Sensing in Escherichia coli. J Biol Chem 2004, 279:8530-8538.
  48. Farmer WR, Liao JC: Improving Lycopene Production in Escherichia coli by Engineering Metabolic Control. Nature Biotechnol 2000, 18:533-537.
  49. Nyström T: The glucose-starvation stimulon of Escherichia coli: induced and repressed synthesis of enzymes of central metabolic pathways and role of acetyl phosphate in gene expression and starvation survival. Mol Microbiol 1994, 12:833-843.
  50. Feng J, Atkinson MR, Mccleary W, Stock JB, Wanner BL, Ninfa AJ: Role of phosphorylated Metabolic Intermediates in the Regulation of Glutamine Synthetase Synthesis in Escherichia coli. J Bacteriol 1992, 174:6061-6070.
  51. Sukmarini L, Shimizu K: Metabolic regulation of Escherichia coli and its glnG and zwf mutants under nitrogen limitation. Biochem Eng J 2010, 48:230-236.
  52. Nandineni MR, Laishram RS, Gowrishankar J: Osmosensitivity associated with insertions in argP (iciA) or glnE in glutamate synthase-deficient mutants of Escherichia coli. J Bacteriol 2004, 186:6391-6399.
  53. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H: Construction of Escherichia coli K-12 in- frame, single gene knockout mutants: the Keio collection. Mol Syst Biol 2006, 2.
  54. Peng L, Shimizu K: Effect of fadR gene knockout on the metabolism of Escherichia coli based on analyses of protein expressions, enzyme activities and intracellular metabolite concentrations. Enzyme Microbiol Technol 2006, 38:512-520.
  55. Kabir MM, Shimizu K: Gene expression patterns for metabolic pathway in pgi knockout Escherichia coli with and without phb genes based on RT- PCR. J Biotechnol 2003, 105:11-31.
  56. Sambrook J, Russell DW: Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 3 2001.
  57. Maurizi RM, Fatima Rasulova: Degradation of L-Glutamate Dehydrogenase from Escherichia coli: allosteric regulation of enzyme stability. Arch Biochem Biophys 2002, 397:206-216.
  58. Kingdon HS, Hubbard JS, Stadtman ER: Regulation of glutamine synthetase. Biochemistry 1968, 7:2136-3142.