Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Molecular cloning and comparative analysis of four β-galactosidase genes from Bifidobacterium bifidum NCIMB41171


Bifidobacterium bifidum NCIMB41171 carries four genes encoding different β-galactosidases. One of them, named bbgIII, consisted of an open reading frame of 1,935 amino acid (a.a.) residues encoding a protein with a multidomain structure, commonly identified on cell wall bound enzymes, having a signal peptide, a membrane anchor, FIVAR domains, immunoglobulin Ig-like and discoidin-like domains. The other three genes, termed bbgI, bbgII and bbgIV, encoded proteins of 1,291, 689 and 1,052 a.a. residues, respectively, which were most probably intracellularly located. Two cases of protein evolution between strains of the same species were identified when the a.a. sequences of the BbgI and BbgIII were compared with homologous proteins from B. bifidum DSM20215. The homologous proteins were found to be differentiated at the C-terminal a.a. part either due to a single nucleotide insertion or to a whole DNA sequence insertion, respectively. The bbgIV gene was located in a gene organisation surrounded by divergently transcribed genes putatively for sugar transport (galactoside-symporter) and gene regulation (LacI-transcriptional regulator), a structure that was found to be highly conserved in B. longum, B. adolescentis and B. infantis, suggesting optimal organisation shared amongst those species.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2


  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

  2. Baumgartner S, Hofmann K, Chiquet-Ehrismann R, Bucher P (1998) The discoidin domain family revisited: New members from prokaryotes and homology-based fold prediction. Protein Sci 7:1626–1631

  3. Bezkorovainy A, Catchpole MR (1989) Biochemistry and Physiology of Bifidobacteria. CRC, Florida

  4. Biery CM, Stewart JF, Stellwagen AE, Raleigh EA, Craig LN (2000) A simple in vitro Tn7-based transposition system with low target site selectivity for genome and gene analysis. Nucleic Acids Res 28(5):1067–1077

  5. Derensy-Dron D, Krzewinski F, Brassart C, Bouquelet S, (1999). β-1,3-Galactosyl-N-acetylhexosamine phosphorylase from Bifidobacterium bifidum DSM 20082: characterisation, partial purification and relation to mucin degradation. Biotechnol Appl Biochem 29:3–10

  6. Dumortier V, Brassart C, Bouquelet S, (1994). Purification and properties of a β-galactosidase from Bifidobacterium bifidum exhibiting transgalactosylation reaction. Biotechnol Appl Biochem 19:341–354

  7. 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-alpha-N-acetylgalactosaminidase from Bifidobacterium longum. J Biol Chem 280 (45):37415–37422

  8. Gardy JL, Laird MR, Chen F, Rey S, Walsh SJ, Ester M, Brinkman FSL (2005) PSORTb v.2.0: Expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis. Bioinformatics 21(5):617–23

  9. Gascell A, Crennell S, Taylor G (1995) The three domains of a bacterial sialidase: A β-propeller, an immunoglobulin module and a galactose binding jelly-roll. Structure 3:1197–1205

  10. Gibson GR, Roberfroid MB (1995) Dietary modulation of the human colonic microbiota: Introducing the concept of prebiotics. J Nutr 125:1401–1412

  11. Goulas KA, Tzortzis G, Gibson RG (2006) Development of a process for the production and purification of α- and β-galactooligosaccharides from Bifidobacterium bifidum NCIMB 41171. Int Dairy J 17(6):648–656

  12. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580

  13. Henrissat B, Bairoch A (1993) New families in the classification of glycosyl hydrolases based on amino acid sequences similarities. Biochem J 293:781–788

  14. Hofmann K, Stoffel W, (1993) TMbase—A database of membrane spanning proteins segments. Biol Chem Hoppe-Seyler 374:166

  15. Hung MN, Xia Z, Hu NT, Lee B (2001) Molecular and biochemical analysis of two β-galactosidases from Bifidobacterium infantis HL96. Appl Environ Microbiol 67 (9):4256–4263

  16. Katayama T, Sakuma A, Kimura T, Makimura Y, Hiratake J, Sakata K, Yamanoi T, Kumagai H, Yamamoto K (2004) Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-L-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95). J Bacteriol 186(15):4885–4893

  17. Kitaoka M, Tian J, Nishimoto M (2005) Novel putative galactose operon involving lacto-N-biose phosphorylase in Bifidobacterium longum. Appl Environ Microbiol 71(6):3158–3162

  18. Lawson PA, Gharbia SE, Shatt HN, Clark DR (1989) Recognition of Fusobacterium nucleatum subgroups FN-1, FN-2 and FN-3 by ribosomal-RNA gene restriction patterns. FEMS Microbiol Lett 65(1–2):41–45

  19. Moller PL, Jorgensen F, Hansen OC, Madsen SM, Stougaard P (2001) Intra-and extracellular β-galactosidases from Bifidobacterium bifidum and B. infantis: Molecular cloning, Heterologous Expression, and Comparative Characterisation. Appl Environ Microbiol 67(5):2276–2283

  20. Navarre WW, Schneewind O (1999) Surface proteins in Gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. Microbiol Mol Biol Review 63:174–229

  21. Nielsen H, Engelbrecht J, Brunak S, Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6

  22. Nquyen CC, Saier MH Jr (1995) Phylogenetic, structural and functional analyses of the LacI-GalR family of bacterial transcription factors. FEBS Lett 377:98–102

  23. Parkinson JS (1993) Signal transduction schemes in bacteria. Cell 73:857–871

  24. Prenosil JE, Stuker E, Bourne JR (1987) Formation of oligosaccharides during enzymatic lactose: Part I: State of the art. Biotechnol Bioeng 30:1019–1025

  25. Reese MG (2001) Application of a time-delay neural network to promoter annotation in the Drosomphila melanogaster genome. Comput Chem 26:51–56

  26. Roger LC, McCartney AL (2005) Prebiotics and the infant microbiota. In: Tannock GW (ed) Probiotics and Prebiotics: Scientific aspects. Caister Academic, UK

  27. Rossi M, Altomare L, Conzalez Vara y Rodriguez P, Brigidi P, Matteuzzi D (2000) Nucleotide sequence, expression and transcriptional analysis of the Bifidobacterium longum MB 219 lacZ gene. Arch Microbiol 174:74–80

  28. Sambrook J, Russell WD (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, New York

  29. Satokari RM, Vaughan EE, Smidt H, Saarela M, Matto J, de Vos WM (2003) Molecular approaches for the detection and identification of bifidobacteria and lactobacilli in the human gastrointestinal tract. System Appl Microbiol 26:572–584

  30. Schell MA, Karmiratzou M, Snel B, Vilanova D, Berger B, Pessi G, Zwahlen MC, Desiere F, Bork P, Delley M, Pridmore DR, Arigoni F (2002) The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract. Proc Natl Acad Sci USA 99(22):14422–14427

  31. Sgorbati B, Biavati B, Palenzona D (1995) The genus of Bifidobacterium: In: Wood BJB and Holzapfel WH (eds) The Lactic Acid Bacteria. Blackie Academic and Professional, London

  32. Suzuki T, Tsuda Y, Kanou N, Inoue T, Kumazaki K, Nagano S, Hirai S, Tanaka K, Watanabe K, (2006) Bifidobacterium adolescentis complete genome sequence. NCBI Database

  33. Tzortzis G, Goulas AK, Gibson GR (2005a) Synthesis of prebiotic galactooligosaccharides using the whole cells of a novel strain Bifidobacterium bifidum NCIMB 41171. Appl Microbiol Biotechnol 68:412–416

  34. Tzortzis G, Goulas AK, Gee MJ, Gibson RG (2005b) A novel galactooligosaccharide mixture increases the bifidobacterial population numbers in a continuous in vitro fermentation system and in the proximal colonic contents of pigs in vivo. J Nutr 135:1726–1731

  35. Van Laere KMJ, Abee T, Schols HA, Beldman G, Voragen AGJ (2000) Characterisation of a novel β-galactosidase from Bifidobacterium adolescentis DSM 20083 active towards transgalactooligosaccharides. Appl Environ Microbiol 66(4):1379–1384

Download references


This work was supported by the Greek State Scholarship’s Foundation (I.K.Y.).

Author information

Correspondence to Theodoros K. Goulas.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Goulas, T.K., Goulas, A.K., Tzortzis, G. et al. Molecular cloning and comparative analysis of four β-galactosidase genes from Bifidobacterium bifidum NCIMB41171. Appl Microbiol Biotechnol 76, 1365–1372 (2007).

Download citation


  • B. bifidum
  • Bifidobacteria
  • Galactosidase
  • Beta-galactosidase
  • Protein evolution