Archives of Microbiology

, Volume 189, Issue 2, pp 157–167 | Cite as

Characterisation of glutamine fructose-6-phosphate amidotransferase (EC and N-acetylglucosamine metabolism in Bifidobacterium

  • Sophie  Foley
  • Emilie  Stolarczyk
  • Fadoua Mouni
  • Colette Brassart
  • Olivier Vidal
  • Eliane Aïssi
  • Stéphane Bouquelet
  • Frédéric Krzewinski
Original Paper


Bifidobacterium bifidum, in contrast to other bifidobacterial species, is auxotrophic for N-acetylglucosamine. Growth experiments revealed assimilation of radiolabelled N-acetylglucosamine in bacterial cell walls and in acetate, an end-product of central metabolism via the bifidobacterial d-fructose-6-phosphate shunt. While supplementation with fructose led to reduced N-acetylglucosamine assimilation via the d-fructose-6-phosphate shunt, no significant difference was observed in levels of radiolabelled N-acetylglucosamine incorporated into cell walls. Considering the central role played by glutamine fructose-6-phosphate transaminase (GlmS) in linking the biosynthetic pathway for N-acetylglucosamine to hexose metabolism, the GlmS of Bifidobacterium was characterized. The genes encoding the putative GlmS of B. longum DSM20219 and B. bifidum DSM20082 were cloned and sequenced. Bioinformatic analyses of the predicted proteins revealed 43% amino acid identity with the Escherichia coli GlmS, with conservation of key amino acids in the catalytic domain. The B. longum GlmS was over-produced as a histidine-tagged fusion protein. The purified C-terminal His-tagged GlmS possessed glutamine fructose-6-phosphate amidotransferase activity as demonstrated by synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. It also possesses an independent glutaminase activity, converting glutamine to glutamate in the absence of fructose-6-phosphate. This is of interest considering the apparently reduced coding potential in bifidobacteria for enzymes associated with glutamine metabolism.


Bifidobacterium GlmS Glutamine fructose-6-phosphate amidotransferase N-acetylglucosamine 


  1. Altschul SF, Madden TL, Schaffer AA, Zhang J, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  2. Alvarez-Anorve LI, Calcagno ML, Plumbridge J (2005) Why does Escherichia coli grow more slowly on glucosamine than on N-acetylglucosamine? Effects of enzyme levels and allosteric activation of GlcN6P deaminase (NagB) on growth rates. J Bacteriol 187:2974–2982PubMedCrossRefGoogle Scholar
  3. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  4. Bustos-Jaimes I, Ramirez-Costa M, De Anda-Aguilar L, Hinojosa-Ocanaand P, Calcagno ML (2005) Evidence for two different mechanisms triggering the change in quartenary structure of the allosteric enzyme, glucosamine-6-phosphate isomerase (deaminase) from Escherichia coli. Biochem 44:1127–1135CrossRefGoogle Scholar
  5. Calcagno M, Campos PJ, Mulliert G, Suastegui M (1984) Purification, molecular and kinetic properties of glucosamine-6-phosphate isomerase (deaminase) from Escherichia coli. Biochim Biophys Acta 787:165–173PubMedGoogle Scholar
  6. Chiang BL, Sheih YH, Wang LH, Liao CK, Gill HS (2000) Enhancing immunity by dietary consumption of a probiotic lactic acid bacterium (Bifidobacterium lactis HN019): optimization and definition of cellular immune responses. Eur J Clin Nutr 54:849–855PubMedCrossRefGoogle Scholar
  7. Derensy-Dron D, Krzewinski F, Brassart C, Bouquelet S (1999) β-1,3-galactosyl-N-acetylhexosamine phosphorylase from Bifidobacterium bifidum DSM 20082: characterization, partial purification and relation to mucin degradation. Biotechnol Appl Biochem 29:3–10PubMedGoogle Scholar
  8. Dutka-Malen S, Mazodier P, Badet B (1988) Molecular cloning and overexpression of the glucosamine synthetase from Escherichia coli. Biochimie 70:287–290PubMedCrossRefGoogle Scholar
  9. Fujita K, Oura F, Nagamine N, Katayama T, Hiratake J, Sakata K, Kumagai H, Yamamoto K (2005) Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo-α-N-acetylgalatosaminidase from B. longum. J Biol Chem 280:37415–37422PubMedCrossRefGoogle Scholar
  10. Good TA, Bessman SP (1964) Determination of glucosamine and galactosamine using borate buffer for modification of the Elson–Morgan and Morgan–Elson reactions. Anal Biochem 9:253–262PubMedCrossRefGoogle Scholar
  11. Gygory P, Kuhn R, Rose CS, Springer GF (1954) Bifidus factor. II. Its occurrence in milk from different species and in other natural products. Arch Biochem Biophys 48:202–208CrossRefGoogle Scholar
  12. Haarman M, Knol J (2005) Quantitative real-time PCR assays to identify and quantify fecal Bifidobacterium species in infants receiving a prebiotic infant formula. Appl Environ Microbiol 71:2318–2324PubMedCrossRefGoogle Scholar
  13. Harmsen HJ, Wildeboer-Veloo AC, Raangs GC, Wagendrop AA, Klijn N, Bindels JG, Welling GW (2000) Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods. J Pediatr Gastroenterol Nutr 30:61–67PubMedCrossRefGoogle Scholar
  14. Katayama T, Fujita K, Yamamoto K (2005) Novel bifidobacterial glycosidases acting on sugar chains of mucin glycoproteins. J Biosci Bioeng 99:457–465PubMedCrossRefGoogle Scholar
  15. Kitaoka M, Tian J, Nishimoto M (2005) Novel putative galactose operon involving lacto-N-biose phosphorylase in Bifidobacterium longum. Appl Environ Microbiol 71:3158–3162PubMedCrossRefGoogle Scholar
  16. Komatsuzawa H, Fujiwara T, Nishi H, Yamada S, Ohara M, McCallum N, Berger-Bächi B, Sugai M (2004) The gate controlling cell wall synthesis in Staphylococcus aureus. Mol Microbiol 53:1221–1231PubMedCrossRefGoogle Scholar
  17. Krzewinski F, Brassart C, Gavini F, Bouquelet S (1997) Glucose and galactose transport in Bifidobacterium bifidum DSM 20082. Curr Microbiol 35:175–179PubMedCrossRefGoogle Scholar
  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of head bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  19. Lambert R, Zilliken F (1965) Novel growth factors for Lactobacillus bifidus var pennsylvanicus. Arch Biochem Biophys 110:544–550PubMedCrossRefGoogle Scholar
  20. Matsuki T, Watanabe K, Fujimoto J, Kado Y, Takada T, Matsumoto K, Tanaka R (2004) Quantitative PCR with 16s rRNA-gene-targeted species-specific primers for analysis of human intestinal bifidobacteria. Appl Environ Microbiol 70:167–173PubMedCrossRefGoogle Scholar
  21. Milewski S (2002) Glucosamine-6-phosphate synthase—the multi-facets enzyme. Biochem Biophys Acta 1597:173–192PubMedGoogle Scholar
  22. Olchowy J, Kur K, Sachadyn P, Milewski S (2006) Construction, purification, and functional characterization of His-tagged Candida albicans glucosamine-6-phosphate synthase expressed in Escherichia coli. Protein Expr Purif 46:309–315PubMedCrossRefGoogle Scholar
  23. Ouwehand AC, Isolauri E, He F, Hashimoto H, Benno Y, Salminen S (2001) Differences in Bifidobacterium flora composition in allergic and healthy infants. J Allergy Clin Immunol 18:144–145CrossRefGoogle Scholar
  24. Ouwehand AC, Isolauri E, Salminen S (2002) The role of intestinal microflora for the development of the immune system in early childhood. Eur J Nutr 41:32–37CrossRefGoogle Scholar
  25. Petschow BW, Talbott RD (1991) Response of Bifidobacterium species to growth promoters in human and cow milk. Pediatr Res 29:208–213PubMedCrossRefGoogle Scholar
  26. Plumbridge JA (1995) Co-ordinated regulation of amino sugar biosynthesis and degradation: the NagC repressor acts as both an activator and a repressor for the transcription of the glmUS operon and requires two separated NagC binding sites. EMBO J 14:3958–3965PubMedGoogle Scholar
  27. Plumbridge JA, Cochet O, Souza JM, Altamirano MM, Calcagno ML, Badet B (1993) Coordinates regulation of amino sugar-synthesizing and -degrading enzymes in Escherichia coli K-12. J Bacteriol 175:4951–4956PubMedGoogle Scholar
  28. Richez C, Boetzel J, Floquet N, Koteshwar K, Stevens J, Badet B, Badet-Denisot M-A (2007) Expression and purification of active human internal His6-tagged l-glutamine:d-Fructose-6P amidotransferase I. Protein Expr Purif 54:45–53PubMedCrossRefGoogle Scholar
  29. Rimington C (1931) Carbohydrate complex of the serum proteins. II. Improved method for isolation and redetermination of glucosaminodiamannose from proteins of ox blood. Biochem J 25:1062–1071PubMedGoogle Scholar
  30. Rossi M, Altomare L, Gonzalez Vara y Rodriguez A, Brigidi P, Matteuzzi D (2000) Nucleotide sequence, expression and transcriptional analysis of the Bifidobacterium longum MB 219 lacZ gene. Arch Microbiol 174:74–80PubMedCrossRefGoogle Scholar
  31. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
  32. Schell MA, Karmirantzou M, Snel B, Vilanova D, Berger B, Pessi G, Zwahlen MC, Desiere F, Bork P, Delley M, Pridmore RD, Arigoni F (2002) The genome sequence of Bifidobacterium longum reflects its adaptation to the gastrointestinal tract. Proc Natl Acad Sci USA 99:14422–14427PubMedCrossRefGoogle Scholar
  33. Skerman VBD, McGowan V, Sneath PHA (1980) Approved lists of bacterial names. Int J Syst Bacteriol 30:225–420CrossRefGoogle Scholar
  34. Studier FW, Rosenberg AH, Dunn JJ (1990) Use of T7 RNA polymerase to direct the expression of cloned genes. Methods Enzymol 185:60–89PubMedCrossRefGoogle Scholar
  35. Teplyakov A, Obmolova G, Badet B, Badet-Denisot M-A (2001) Channeling of ammonia in glucosamine-6-phophate synthase. J Mol Biol 313:1093–1102PubMedCrossRefGoogle Scholar
  36. Veerkamp JH (1969) Uptake and metabolism of determinatives of 2-deoxy-2-amino-d-glucose in Bifidobacterium bifidum var. pennsylvanicus. Arch Biochem Biophys 129:248–256PubMedCrossRefGoogle Scholar
  37. Vogler AP, Trentmann S, Lengeler JW (1989) Alternative route for biosynthesis of amino sugars in Escherichia coli K-12 mutants of a catabolic isomerase. J Bacteriol 171:6585–6592Google Scholar
  38. Weingand-Ziade A, Gerber-Decombaz C, Affolter M (2003) Functional characterization of a salt- and thermotolerant glutaminase from Lactobacillus rhamnosus. Enzyme Microb Technol 32:862–86CrossRefGoogle Scholar
  39. Yamashita T, Ashiuchi M, Ohnishi K, Kato S, Nagata S, Misono H (2004) Molecular identification of monomeric aspartate racemase from Bifidobacterium bifidum. Eur J Biochem 271:4798–4803PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Sophie  Foley
    • 1
  • Emilie  Stolarczyk
    • 2
    • 3
  • Fadoua Mouni
    • 2
  • Colette Brassart
    • 2
  • Olivier Vidal
    • 2
  • Eliane Aïssi
    • 2
  • Stéphane Bouquelet
    • 2
  • Frédéric Krzewinski
    • 2
  1. 1.School of Life SciencesNapier UniversityEdinburghUK
  2. 2.Unité de Glycobiologie Structurale et Fonctionnelle, UMR CNRS-USTL 8576, IFR147Université des Sciences et Technologies de LilleVilleneuve d’AscqFrance
  3. 3.Université Pierre et Marie Curie-Paris6ParisFrance

Personalised recommendations