Abstract
A chromosomal DNA fragment of 7.8 kb from Lactobacillus coryniformis CECT 5711 was cloned in Escherichia coli K-12 and was found to express a functional β-galactosidase. Nucleotide sequence analysis showed that this fragment contained two partially overlapping genes, the lacL (1,881 bp) and the lacM (960 bp), that encode the subunits of a heterodimeric β-galactosidase, with estimated molecular masses of 72,129 and 35,233 Da, respectively. Other three incomplete open reading frames showing homology to another β-galactosidase, an α-galactosidase, and a galactokinase, respectively, were also found. The L. coryniformis β-galactosidase was overproduced in E. coli by using an isopropyl-β-d-thiogalactopyranoside (IPTG) expression system. Two new proteins with an estimated M r s of approximately 72,000 and 35,000 appeared upon induction with IPTG, and extracts of the recombinant E. coli strain showed β-galactosidase activity.
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References
Axelson L (1998) Lactic acid bacteria: classification and physiology. In: Salminen S, Von Wright A (eds) Lactic acid bacteria: microbiology and functional aspects. Marcel Dekker, New York, NY, pp 1–71
Bradford MM (1976) A rapid method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254
David S, Stevens H, van Riel M, Simons G, de Vos WM (1992) Leuconostoc lactis β-galactosidase is encoded by two overlapping genes. J Bacteriol 174:4475–4481
De Vrese M, Stegelmann A, Richter B, Fenselau S, Laue C, Schrezenmeir J (2001) Probiotics—compensation for lactase insufficiency. Am J Clin Nutr 73S:421–429
Fernández M, Margolles A, Suárez JE, Mayo B (1999). Duplication of the β-galactosidase gene in some Lactobacillus plantarum strains. Int J Food Microbiol 48:113–123
Fortina MG, Ricci G, Mora D, Guglielmetti S, Manachini PL (2003) Unusual organization for lactose and galactose gene clusters in Lactobacillus helveticus. Appl Environ Microbiol 69:3238–3243
Fuller R (1989) Probiotics in man and animals. J Appl Bacteriol 66:365–378
Iida S, Marcoli R, Bickle A (1982) Phenotypic reversion of an IS1-mediated deletion mutation: a combined role for point mutations and deletions in transposon evolution. EMBO J 1:755–759
Kleerebezem M, Boekhorst J, van Kranenburg R, Molenaar D, Kuipers OP, Leer R, Tarchini R, Peters SA, Sandbrink HM, Fiers MW, Stiekema W, Klein Lankhorst RM, Bron PA, Hoffer SM, Groot MN, Kerkhoven R, de Vries M, Ursing B, de Vos WM, Siezen RJ (2004) Complete genome sequence of Lactobacillus plantarum WCFS1. Proc Natl Acad Sci U S A 100:1990–1995
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Leong-Morgenthaler PM, Zwahlen MC, Hottinger H (1991) Lactose metabolism in Lactobacillus bulgaricus: analysis of the primary structure and expression of the genes involved. J Bacteriol 173:1951–1957
Martin R, Olivares M, Marín ML, Xaus J, Fernández L, Rodríguez JM (2005) Characterization of a reuterin-producing Lactobacillus coryniformis strain isolated from goat’s milk cheese. Int J Food Microbiol 104:267–277
Platt T (1986) Transcription termination and its regulation of gene expression. Annu Rev Biochem 55:339–372
Poolman B, Royer TJ, Mainzer SE, Schmidt BF (1989) Lactose transport system of Streptococcus thermophilus: a hybrid protein with homology to the melibiose carrier and enzyme III of the phosphoenolpyruvate-dependent phosphotransferase system. J Bacteriol 171:244–253
Pridmore RD, Berger B, Desiere F, Vilanova D, Barretto C, Pittet AC, Zwahlen MC, Rouvet M, Altermann E, Barrangou R, Mollet B, Mercenier A, Klaenhammer T, Arigoni F, Schell MA (2004) The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533. Proc Natl Acad Sci U S A 101:2512–2517
Salminen S, Bouley C, Boutron-Rualt M-C, Cummings JH, Franck A, Gibson GR, Isolauri E, Moreau MC, Roberfroid MB, Rowland IR (1998) Functional food science and gastrointestinal physiology and function. Br J Nut 80S:147–171
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Laboratory Press, Cold Spring Harbor, NY
Schachtsiek M, Hammes WF, Hertel C (2004) Characterization of Lactobacillus coryniformis DSM 20001T surface protein Cpf mediating coaggregation with and aggregation among pathogens. Appl Environ Microbiol 70:7078–7085
Shimizu-Kadota M, Flickinger JL, Chassy BM (1988) Evidence that Lactobacillus casei insertion element ISL1 has a narrow host range. J Bacteriol 170:4976–4978
Silvestroni A, Connes C, Sesma F, de Giori GS, Piard JC (2002) Characterization of the melA locus for α-galactosidase in Lactobacillus plantarum. Appl Environ Microbiol 68:5464–5471
Vaillancourt K, Moineau S, Frenette M, Lessard C, Vadeboncoeur C (2002) Galactose and lactose genes from the galactose-positive bacterium Streptococcus salivarius and the phylogenetically related galactose-negative bacterium Streptococcus thermophilus: organization, sequence, transcription, and activity of the gal gene products. J Bacteriol 184:785–793
Vaughan EE, David S, de Vos W (1996) The lactose transporter in Leuconostoc lactis is a new member of the LacS subfamily of galactoside-pentose-hexuronide translocators. Appl Environ Microbiol 62:1574–1582
Xaus J, Martín R, Rodríguez JM, Boza J, Jiménez J (2004) Probiotic strains, a process for the selection of them, compositions thereof, and their use. Patent application WO 2004/003235
Acknowledgements
We thank A. Carceles, I. Sánchez, and C. Membrilla for their skillful technical assistance and L. Fernandez, M. Valdivieso, and C. Ronchel for their helpful discussion of the results. José M. Corral received a research fellowship from Fundación Universidad-Empresa (University of Granada) and Puleva Biotech SA.
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Corral, J.M., Bañuelos, O., Adrio, J.L. et al. Cloning and characterization of a β-galactosidase encoding region in Lactobacillus coryniformis CECT 5711. Appl Microbiol Biotechnol 73, 640–646 (2006). https://doi.org/10.1007/s00253-006-0510-7
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DOI: https://doi.org/10.1007/s00253-006-0510-7