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Applied and Environmental Microbiology, July 1999, p. 3008-3014, Vol. 65, No. 7
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Biochemical and Structural Characterization of the Glucan and Fructan Exopolysaccharides Synthesized by the Lactobacillus reuteri Wild-Type Strain and by Mutant Strains

G. H. Van Geel-Schutten,1 E. J. Faber,2 E. Smit,1 K. Bonting,3 M. R. Smith,4 B. Ten Brink,1 J. P. Kamerling,2 J. F. G. Vliegenthart,2 and L. Dijkhuizen3,*

TNO Nutrition and Food Research, Department of Microbiology, 3700 AJ Zeist,1 Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, NL-3508 TB Utrecht,2 Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, 9751 NN Haren,3 and Department of Microbiology, NIZO, 6710 BA Ede,4 The Netherlands

Received 7 January 1999/Accepted 14 April 1999

Lactobacillus reuteri LB 121 cells growing on sucrose synthesize large amounts of a glucan (D-glucose) and a fructan (D-fructose) with molecular masses of 3,500 and 150 kDa, respectively. Methylation studies and 13C or 1H nuclear magnetic resonance analysis showed that the glucan has a unique structure consisting of terminal, 4-substituted, 6-substituted, and 4,6-disubstituted alpha -glucose in a molar ratio of 1.1:2.7:1.5:1.0. The fructan was identified as a (2right-arrow6)-beta -D-fructofuranan or levan, the first example of levan synthesis by a Lactobacillus species. Strain LB 121 possesses glucansucrase and levansucrase enzymes that occur in a cell-associated and a cell-free state after growth on sucrose, raffinose, or maltose but remain cell associated during growth on glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sucrose culture supernatants, followed by staining of gels for polysaccharide synthesizing activity with sucrose as a substrate, revealed the presence of a single glucansucrase protein of 146 kDa. Growth of strain LB 121 in chemostat cultures resulted in rapid accumulation of spontaneous exopolysaccharide-negative mutants that had lost both glucansucrase and levansucrase (e.g., strain K-24). Mutants lacking all levansucrase activity specifically emerged following a pH shiftdown (e.g., strain 35-5). Strain 35-5 still possessed glucansucrase and synthesized wild-type glucan.

* Corresponding author. Mailing address: Department of Microbiology, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands. Phone: (31) 50-363-2153. Fax: (31) 50-363-2154. E-mail: L.Dijkhuizen{at}biol.rug.nl.

Applied and Environmental Microbiology, July 1999, p. 3008-3014, Vol. 65, No. 7
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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