1 Centre for Carbohydrate Bioengineering (CCB), TNO-University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands
2 Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands
3 Innovative Ingredients and Products Department, TNO Nutrition and Food Research, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands
4 Innovative Ingredients and Products Department, TNO Nutrition and Food Research, Rouaanstraat 27, 9723 CC, Groningen, The Netherlands
Correspondence
L. Dijkhuizen
L.Dijkhuizen{at}biol.rug.nl
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences of the Lactobacillus gtf genes gtf180, gtfML1, gtf33, gtfKg3, gtfKg15 and gtfB and their flanking regions are AY697430, AY697431, AY697432, AY697433, AY697434 and AY697435.
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INTRODUCTION |
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The distribution of glucosidic linkages has been elucidated for the glucans synthesized by heterologously produced GTF enzymes including: (i) 13 GTFs from seven Streptococcus strains (Monchois et al., 1999c; Hanada et al., 2002
; Konishi et al., 1999
), (ii) seven GTFs from four Leuconostoc strains (Monchois et al., 1999c
; Bozonnet et al., 2002
; Neubauer et al., 2003
; Funane et al., 2001
; Arguello-Morales et al., 2000
) and (iii) from Lb. reuteri strain 121 (Kralj et al., 2002
). Only gtf genes encoding either dextran- or mutan-sucrase enzymes have been characterized in the genus Streptococcus (Monchois et al., 1999c
; Hanada et al., 2002
; Konishi et al., 1999
). Leuconostoc strains carry gtf genes encoding mainly dextransucrase enzymes, but an alternansucrase-encoding gene and one gene encoding a glucansucrase that synthesizes large amounts of
-(1
2)-branch linkages have also been characterized (Arguello-Morales et al., 2000
; Bozonnet et al., 2002
; Monchois et al., 1999c
).
The GTF enzymes of Streptococcus species are generally produced constitutively (Kim & Robyt, 1994). GTF enzymes of Leuconostoc species are specifically induced by sucrose. This is disadvantageous for several applications, and therefore some constitutive mutants were constructed (Kim & Robyt, 1994
; Kitaoka & Robyt, 1998
). Reuteransucrase from Lb. reuteri 121 is produced constitutively (van Geel-Schutten et al., 1999
). The GTF enzymes from Lb. reuteri 180 and Lb. parabuchneri 33 are also produced constitutively (unpublished data).
The only streptococcal species that is associated with food technology is Streptococcus thermophilus, which is used in the manufacture of yoghurt. The genus Streptococcus contains several well-known pathogens (e.g. Streptococcus pneumoniae) (Axelsson, 1998; Leroy & De Vuyst, 2004
). Furthermore, glucans produced by oral streptococci play a key role in the cariogenesis process, by enhancing the attachment and colonization of cariogenic bacteria (Loesche, 1986
). Leuconostoc strains play an important role in vegetable fermentations (Axelsson, 1998
; Leroy & De Vuyst, 2004
).
Lactobacilli are widespread in nature, and many species have found applications in the food industry (e.g. dairy products, sourdough) (Axelsson, 1998; De Vuyst & Degeest, 1999
). Several Lb. reuteri strains are able to produce antimicrobial metabolites (e.g. reutericyclin, reuterin and reutericin), which delay the growth of some food-borne pathogens (Kabuki et al., 1997
; Ganzle et al., 2000
; Talarico et al., 1988
). Furthermore, some Lb. reuteri strains have probiotic properties, as has been demonstrated in humans and various animals (Casas et al., 1998
; Valeur et al., 2004
). The range of glucans and oligosaccharides produced by GTF enzymes present in lactobacilli (Kralj et al., 2004
) may potentially act as prebiotics by stimulating the growth of probiotic strains or of beneficial endogenic strains of the gastrointestinal tract (Monsan & Paul, 1995
; Olano-Martin et al., 2000
; Chung & Day, 2002
).
Lactobacillus reuteri strains producing glucans thus possess the following general advantages: (i) constitutive GTF enzyme production, (ii) safe (GRAS status) and (iii) potential pro- and prebiotic properties. Glucans and glucooligosaccharides from lactobacilli are therefore interesting and feasible alternatives to the additives currently used in the production of foods (e.g. sourdough, yoghurts, health foods). Although different Lactobacillus strains are able to produce glucans (Kralj et al., 2003; Tieking et al., 2003
; Sidebotham, 1974
; van Geel-Schutten et al., 1998
), only the Lb. reuteri GTFA enzyme has been characterized thus far. This enzyme synthesizes a highly branched glucan (reuteran) containing
-(1
4) and
-(1
6) linkages (Kralj et al., 2002
). The same types of glucosidic linkages were synthesized in its oligosaccharide products (Kralj et al., 2004
). Recently, we have shown that lactobacilli, in fact, contain DNA sequences of other putative glucansucrase genes (Kralj et al., 2003
).
This paper describes the characterization of the glucans produced by six different Lactobacillus strains, and the isolation and characterization of the corresponding gtf genes and GTF enzymes. The data show that members of the genus Lactobacillus contain the same variety of gtf genes, GTF enzymes and glucan products as Leuconostoc and Streptococcus species.
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METHODS |
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Isolation of DNA.
Lactobacillus total DNA was isolated according to Nagy et al. (1995). Plasmid DNA of E. coli was isolated using the alkaline lysis method (Birnboim & Doly, 1979
) or with a Wizard Plus SV plasmid extraction kit (Promega).
Molecular techniques.
General procedures for gene cloning, E. coli DNA transformations, DNA manipulations, and agarose gel electrophoresis were as described by Sambrook et al. (1989). Restriction endonuclease digestions and ligations with T4 DNA ligase were performed as recommended by the enzyme suppliers (New England Biolabs; Roche Biochemicals). Primers were obtained from Eurogentec. Sequencing was performed by GATC. DNA was amplified by PCR on a DNA Thermal Cycler PTC-200 (MJ Research) using Pwo DNA polymerase (Roche Biochemicals) or Expand High Fidelity polymerase (Roche Biochemicals). For inverse PCR (iPCR) the Expand High Fidelity PCR system (Roche Biochemicals) was used as described by the supplier. Fragments were isolated from agarose gels using a Qiagen gel extraction kit following the instructions of the supplier.
Identification and nucleotide sequence analysis of the glucansucrase genes.
A first fragment of the glucansucrase genes was isolated by PCR amplification of chromosomal DNA from the different Lactobacillus strains using degenerate primers (DegFor and DegRev) which were based on sequence similarity between conserved regions, located in the catalytic core, of different GTF enzymes of Gram-positive bacteria (Kralj et al., 2003). The
660 bp amplified fragments were used to identify appropriate restriction sites and to design primers for subsequent iPCR reactions (Triglia et al., 1988
) (Table 1
).
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Enzyme assays.
His-tag-purified GTF enzymes from E. coli extracts and culture supernatants of Lactobacillus strains grown on MRSs were used as a source of enzyme for the enzyme assays. GTF total activity was measured by determining the release of fructose from sucrose at 37 °C in 50 mM sodium acetate buffer (pH 5·5) containing 1 mM CaCl2 and 100 mM sucrose (van Geel-Schutten et al., 1999).
SDS-PAGE followed by activity staining.
Gel electrophoresis and GTF activity staining of gels with periodic acid Schiff reagents was performed as described previously (Kralj et al., 2003).
Production of glucans and analysis
(i) Polymer production.
Glucans synthesized by cultures of the Lactobacillus strains and glucans synthesized by the His-tag-purified GTF enzymes from E. coli were produced and isolated by ethanol precipitation as described previously (Kralj et al., 2002).
(ii) Methylation.
Polysaccharides were permethylated using methyl iodide and dimsyl sodium (-Na+) in DMSO at room temperature (Hakomori, 1964
). After hydrolysis with 2 M trifluoroacetic acid (1 h, 125 °C), the partially methylated monosaccharides were reduced with NaBD4 (Harris et al., 1984
). Mixtures of partially methylated alditol acetates obtained were analysed by GLC on a CP Sil 5 CB column (25 mx0·53 mm; Chrompack) and by GLC- MS on an RTX 5 Sil MS (30 mx0·25 mm; Restek) column (Chaplin, 1982
; Jansson et al., 1976
).
(iii) Molecular masses of the glucans.
Molecular mass analysis was performed as described previously, using high-performance size-exclusion chromatography (HPSEC) coupled on-line with multi-angle laser light scattering (MALLS) and differential refractive index detection (Kralj et al., 2002).
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RESULTS AND DISCUSSION |
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The gtf genes in the different Lactobacillus species/strains appear to have different chromosomal locations, with a relatively high frequency of transposase homologues flanking the different gtf genes (Fig. 1, Table 3
).
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N-terminal variable regions
The protein structures of GTFB, GTF180 and GTFML1 found in three different Lb. reuteri strains were very similar to that of GTFA of Lb. reuteri 121 (Fig. 2). All three GTFs contained a relatively large and highly similar variable region (
700 amino acids) with 5 RDV repeats (sequence R(P/N)DV-x12-SGF-x1922-R(Y/F)S, where x represents a non-conserved amino acid residue), as previously observed in GTFA of Lb. reuteri 121 (Kralj et al., 2002
) (Fig. 2
). The variable domains of the other three isolated GTFs were smaller and contained different repeating units from the Lb. reuteri GTFs. In their variable region, GTFKg3 and GTFKg15 contained, respectively, five and six conserved and less-conserved YG-repeats (sequence NDGYYFxxxGxxH°x(G/N)H°H°H°, where x represents a non-conserved amino acid residue and H° represents a hydrophobic amino acid residue) (Fig. 2
) (Giffard & Jacques, 1994
). In the variable region of GTF33, nine short unique repeating units, designated TTQ, were found. These repeats were 15 amino acids long, with TTTQN(A/T)(P/A)NN(S/G)N(D/G)PQS as their sequence, and showed no significant similarity to any protein motif present in databases (Fig. 2
). Different repeating units also could be identified in the N-terminal variable domain of other glucansucrases: A-repeats in alternansucrase and dextransucrases of Ln. mesenteroides (Janecek et al., 2000
); motif T, TDDKA(A/T)TTA(A/D)TS (bold type indicates conserved amino acids) in DSRT of Ln. mesenteroides NRRL B-512F (Funane et al., 2000
); motif S, PA(A/T)DKAVDTTP(A/T)T, bold type indicates conserved amino acids) in DSRE of Ln. mesenteroides NRRL B-1299 (Bozonnet et al., 2002
) and RDV repeats in GTFA of Lb. reuteri 121) (Kralj et al., 2002
). However, deletion studies of the variable domain showed that it did not determine the type of glucosidic linkages present or the size (only determined for GTFA) of the synthesized glucans (Monchois et al., 1999a
; Kralj et al., 2004
).
Catalytic domains
The catalytic domains of the putative GTF enzymes range in size from 741 to 774 amino acids (Fig. 2). Within all the catalytic domains the three completely conserved amino acids already identified as being essential for enzymic activity in other GTF enzymes (Asp1024, Glu1061 and Asp1133; GTFA Lb. reuteri 121 numbering) could be identified (Devulapalle et al., 1997
; Kralj et al., 2003
; Kralj et al., 2004
).
Putative glucan-binding domains (GBDs)
The C-terminal domain of Streptococcus and Leuconostoc GTF enzymes consists of a series of different tandem repeats, which have been divided into four classes: A, B, C and D repeats. Within the AD repeats, a repeating unit designated YG can be distinguished (Giffard & Jacques, 1994). GTFB, GTFML1 and GTF180 possessed a relatively short GBD of 134263 amino acids, comparable with the GBD from GTFA of Lb. reuteri 121 (Fig. 2
) and consisting of several conserved and less-well-conserved YG-repeats (Kralj et al., 2002
). Characterization of sequential C-terminal deletion mutants of GTFA revealed that the C-terminal domain has an important role in glucan binding (Kralj et al., 2004
).
The putative GBDs of the other isolated GTFs were approximately twice as large as the Lb. reuteri GBDs. They contained a varying number of conserved and less-well-conserved YG-repeating units (Fig. 2) and no A, B, C or D repeats could be identified. GTF33 contained, besides the 17 YG-repeats, two unique repeating units, designated KYQ [49 amino acids, AVK(T/A)A(K/Q)(A/T)(Q/K)(L/V)(A/N)K(T/A)KAQ(I/V)(A/T)KYQKALKKAKTTKAK(A/T)QARK(S/N)LKKA(E/N)(T/S)S(F/L)(S/T)KA] that showed no significant similarity to any protein motif present in databases. GTFKg15 possessed, at the end of its putative GBD, an additional stretch which shows similarity to part of a putative extracellular matrix binding protein from Streptococcus pyogenes M1 (AE006525; 44 % similarity and 56 % identity within 75 amino acids) (Fig. 2
).
Expression of the gtf genes in E. coli
Based on the nucleotide sequence information obtained, six different gtf genes were cloned and expressed in E. coli (Table 2). The gtfB gene was expressed as a full-length protein. The gtf180 and gtf33 genes were expressed as proteins with and without their N-terminal variable regions. The gtfML1, gtfKg3 and gtfKg15 genes were expressed as proteins without their N-terminal variable regions (Table 2
). Except for GTFB from Lb. reuteri 121, the His-tag-purified GTF proteins all showed enzymic activity, as measured by fructose release from sucrose. SDS-PAGE showed that, in all cases, protein was present as a band corresponding to the molecular mass of the different truncated and full-length enzymes (data not shown). Staining with Schiff reagents for polymer-synthesizing activity was positive, under the conditions examined, for all recombinant enzymes (except for GTFB, data not shown).
In the supernatant of Lb. reuteri 121 cultures only the GTFA enzyme was found (under the growth conditions tested). Furthermore, the glucan synthesized by Lb. reuteri 121 is identical to the glucan produced by GTFA (Kralj et al., 2002). This suggests that GTFB of Lb. reuteri 121 either synthesizes a similar glucan product or (most likely) is not active under the growth conditions tested. GTFB has a relatively small GBD, compared to GTFA (Fig. 2
). However, deletion studies with GTFA showed that this enzyme was still active after truncation of the GBD to the size (6 YG repeats deleted) of the GBD in GTFB (Kralj et al., 2004
). GTFB also possesses the three catalytically important residues (Devulapalle et al., 1997
; Kralj et al., 2004
). Its inactivity may be caused by the aberrant amino acid sequence at the start of its catalytic core. The highly conserved motif INGQYY, indicating the start of the catalytic core in GTF enzymes, is absent in GTFB. This GTFB region also contains many gaps and overall very poor similarity when compared to other GTF enzymes. Truncations in this region of GTFI from Streptococcus downei Mfe28 resulted in drastic loss of enzyme activity (Monchois et al., 1999b
).
GTFML4 showed high similarity with GTFB of Lb. reuteri 121 (Table 3), including the differences with other GTF enzymes listed above. Furthermore, the organization of gtfML1 and gtfML4 on the genome of Lb. reuteri ML1 was similar to that of gtfA and gtfB from Lb. reuteri 121 (Fig. 1
). Consequently, no further efforts were made to clone the full-length gtfML4 gene. The identical nature of the glucans produced by Lb. reuteri ML1 and by the purified recombinant GTFML1 was confirmed by methylation analysis (Table 4
). It appears likely that under the conditions tested gtfML4 also is not active, as is the case with GTFB.
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Conclusions
This paper reports the first examples, we believe, of isolation and characterization of dextransucrase and mutansucrase genes/enzymes, and dextran/mutan products from Lactobacillus species. The genus Lactobacillus thus contains the same variety of gtf genes, GTF enzymes and glucan products as found within the genera Leuconostoc and Streptococcus, plus the ability to synthesize reuteran (reuteransucrase of Lb. reuteri 121). GTFA, GTF180 and GTFML1 are highly similar (Table 3) but synthesize glucans with different glucosidic linkages. Thus these enzymes are very interesting candidates for structure/function studies aiming to identify amino acid residues responsible for glucosidic-bond specificity.
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Received 11 May 2004;
revised 28 July 2004;
accepted 2 August 2004.
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