INTRODUCTION

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Since they were first discovered by Tissier (33), the bifidobacteria have been investigated extensively by several scientists (e.g., references 23 and 27). In recent years, bifidobacteria have attracted particular attention due to their promising health-promoting properties, for example, reduction of harmful bacteria and toxic compounds in the intestine, prevention of dental caries, reduction of total cholesterol and lipid in serum, and relief of constipation (2, 5, 10, 17, 36, 41). Therefore, live probiotic bifidobacteria, which may improve the microbial balance of the human gastrointestinal tract, have been used to supplement dairy products for many years. Another approach to increase the number of beneficial bacteria in the human intestine is to selectively stimulate their growth by supplementing food with ingredients which can only be metabolized by such bacteria. Certain oligosaccharides, the so-called prebiotics, have been shown to exert this growth-stimulating effect on probiotic bacteria, including bifidobacteria.

So far, most of the probiotic bacteria and the prebiotic oligosaccharides have been used in combination with dairy products, and since these products often contain large amounts of lactose, much attention has been focused on the enzyme -galactosidase (EC 3.2.1.23), which is involved in the bacterial metabolism of lactose. In addition to normal hydrolysis of the -D-galactoside linkage in lactose, some -D-galactosidase enzymes may catalyze the formation of galactooligosaccharides through transfer of one or more D-galactosyl units onto the D-galactose moiety of lactose. This transgalactosylation reaction (12) has been shown to be a characteristic of -galactosidase enzymes from a great variety of bacterial and fungal species (7, 19, 21, 40).

Galactooligosaccharides produced from lactose by transgalactosylation specifically stimulate growth of bifidobacteria (39), and recently Van Laere et al. (37) have described a novel -galactosidase from Bifidobacterium adolescentis that preferentially hydrolyzes galactooligosaccharides. Therefore, it is generally accepted that a structural and catalytic characterization of the -galactosidase enzymes of probiotic bacteria is of central importance for an understanding of their health-promoting effects.

Lactose hydrolysis and transgalactosylation properties of the enzyme have been studied in several probiotic bacteria including bifidobacteria (6, 7, 25, 26, 30, 35, 37), but so far only one DNA sequence of a bifidobacterial -galactosidase gene has been published (Bifidobacterium longum; EMBL accession no. AJ242596) (24), and another sequence has been deposited in a database (Bifidobacterium breve; EMBL accession no. E05040). In this paper, we describe the molecular cloning, sequencing and characterization of three different -galactosidase enzymes from Bifidobacterium bifidum (DSM20215) and one enzyme from Bifidobacterium infantis (DSM20088).

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and culture conditions. Fifteen different bifidobacterial strains were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, and analyzed for their ability to synthesize galactooligosaccharides. Two strains, B. bifidum DSM20215 and B. infantis DSM20088, which were able to synthesize oligosaccharides, were selected for this study. The strains were grown anaerobically using TPY medium (28) at 37°C with BBL GasPak Anaerobic systems (Becton Dickinson and Co., Cockeysville, Md.). DNA cloning was performed using the Escherichia coli strains (i) MT102, a derivative of MC1000 (hsdR-K12) (4), (ii) XL-1-5, an F derivative of XL1-Blue (3), and (iii) ER1458 (22). The E. coli strains were grown in Luria-Bertani (LB) medium (18) supplemented with 100 µg of ampicillin/ml and 40 µg of 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal)/ml when appropriate. The plasmid pBluescript KS() (Stratagene Cloning Systems, La Jolla, Calif.) was the vector used for DNA fragment cloning.

Chemicals and enzymes. Chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.). Restriction enzymes and other enzymes used for DNA manipulation were from New England Biolabs, Inc. (Beverly, Mass.) and were used according to the instructions of the manufacturer.

Isolation of -galactosidase genes. Chromosomal DNA was prepared from a cell pellet harvested from 500 ml of TPY culture. The cells were resuspended in 4.4 ml of lysis solution (20 mM Tris-HCl, 20 mM MgCl₂, 20% glucose, 5 mg of lysozyme/ml, and 350 U of mutanolysin/ml [pH 6.5]) and incubated at 37°C for 60 min. Cells were lysed by the addition of 12 ml of TEN buffer (100 mM Tris-HCl, 1 mM EDTA, and 100 mM NaCl [pH 7.5]), 1.6 ml of 10% sodium dodecyl sulfate (SDS), 1.6 ml of 0.5 M EDTA (pH 7), and 0.3 ml of proteinase K (20 mg/ml), followed by incubation at 37°C for 60 min. Five milliliters of phenol and 5 ml of chloroform were added, and the extraction was repeated until the water phase could easily be separated from the interphase. The genomic DNA was precipitated with isopropanol, resuspended in 10 mM Tris-HCl-1 mM EDTA (pH 8.0), and treated with RNase. The genomic DNA was then digested with restriction enzymes, ligated into pBluescript KS(), digested with the same enzymes, and treated with alkaline phosphatase. Digestion of B. bifidum genomic DNA was performed using BamHI, EcoRI, HindIII, PstI, SacI, KpnI, ApaI, and SalI, whereas B. infantis DNA was digested with KpnI. Ligation mixtures were used to transform E. coli MT102, and -galactosidase-producing clones were identified as blue colonies on X-Gal-containing plates.

Preparation of cell lysates. E. coli cells harboring the recombinant -galactosidase genes were lysed with a French pressure cell. Harvested cells from a 750-ml culture of E. coli ER1458 (optical density at 450 nm = 1) were washed with 50 ml of 50 mM sodium phosphate-10 mM MgCl₂ (pH 6.8) and then resuspended in 7 ml of the same buffer. The French pressure cell was operated at 196 MPa. Alternatively, the cells were resuspended in Z buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, and 50 mM -mercaptoethanol [pH 7.0]), mixed with glass beads (33% [vol/vol], 212 to 300 µm; Sigma), and lysed in an ultrasonic bath by incubation twice for 5 min. Cell debris was removed by centrifugation, and the supernatant was used directly for enzyme activity measurements and enzyme characterization.

Assays for -galactosidase activity. Hydrolysis of o-nitrophenyl (ONP)--D-galactopyranoside at 37°C and pH 7.0 followed by measurement of absorbance at 420 nm was used for determination of -galactosidase activity (18). Assays were performed with Z buffer (for -galactosidases BIF1, BIF2, and INF1) or Z buffer containing 0.5% Triton X-100 (for BIF3), and the reactions were stopped by the addition of 1 M Na₂CO₃. Transgalactosylation assays were performed with 0.4 M lactose, 50 mM Na citrate, and 100 mM Na₂HPO₄ (pH 6.0), and the 50-µl reaction volumes were incubated for approximately 20 h at 40°C. A 5-min incubation at 95°C was used to stop the enzyme reaction. The reaction mixtures were analyzed by thin-layer chromatography on Silica Gel 60 plates (Merck) in a solvent containing butanol, 2-propanol, and water (3:12:4). Samples of 1 µl of diluted sample (1:1 dilution in water) were subjected to three runs. After being dried, the sugars were visualized by spraying with an orcinol reagent, followed by incubation at 100°C for 5 to 10 min.

Molecular mass determination. The native molecular mass of -galactosidases expressed in E. coli was determined by analytical gel filtration on a Superdex 200 HR 10/30 column (Pharmacia) followed by -galactosidase assay of collected fractions. The molecular mass markers used for calibration of the column were thyroglobulin (669 kDa), ferritin (440 kDa), human immunoglobulin G (160 kDa), transferrin (81 kDa), and ovalbumin (43 kDa). The molecular mass of the -galactosidase subunits was determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (15). Samples were reduced with dithiothreitol and loaded onto 7.5 or 10% minigels which were stained with Coomassie brilliant blue or silver stained.

Enzyme purification. The BIF2 -galactosidase was purified from E. coli extract by anion-exchange chromatography on a 5-ml HiTrap Q column (Pharmacia) at pH 7.5, followed by gel filtration on a Sephacryl S-200 HR column (Pharmacia, 1.6 by 60 cm). The INF1 -galactosidase was purified from E. coli extract by gel filtration on a Superdex 200 HR 10/30 column (Pharmacia) followed by anion-exchange chromatography at pH 7.5 on a Mini-Q column (Pharmacia). The BIF3 -galactosidase was purified from E. coli extract by gel filtration on a Superdex 200 HR 10/30 column, run on a SDS-7.5% polyacrylamide gel as described above, transferred to polyvinylidene difluoride (PVDF) membrane (Immobilon P; Millipore), and stained with Coomassie brilliant blue. Digestion of BIF3 polypeptide bound to PVDF with endoproteinase Lys-C and extraction of the resultant proteolytic peptides were performed as described (8). The peptide fragments were purified by reversed-phase chromatography on a SMART system (Pharmacia) equipped with a µRPC C2/C18 SC2.1/10 column (Pharmacia) using a gradient of 0 to 80% acetonitrile in 0.1% trifluoroacetic acid. Peptide sequencing was performed as described below. The expression level of BIF1 was too low to permit purification. Therefore, crude cell extract containing the BIF1 enzyme was used to determine the molecular weight by gel filtration.

N-terminal amino acid sequence analysis. Enzyme samples were run on SDS-polyacrylamide gels as above, transferred to PVDF membrane (Problott; PE Biosystems), stained with Coomassie brilliant blue, and analyzed by Edman degradation with a protein microsequencer (Procise; PE Biosystems).

DNA sequence analysis. DNA sequencing was carried out using Cy-5-labeled primers. Vector specific primers, T3 and T7, were used in the first sequencing reaction mixture, followed by reactions with sequence-specific primers. The reaction mixtures were run with an ALF Express sequencer (Pharmacia). Databases were searched for homologous proteins with the BLAST facility (1). Comparison of amino acid sequences was performed as a BestFit analysis with the Wisconsin software package, version 10.0 (Genetics Computer Group, Madison, Wis.). The gap creation and gap extension penalty parameters for the BestFit analysis were 8 and 2, respectively. Alignment of -galactosidase protein sequences was performed with the ClustalX program (34). The aligned sequences were subsequently imported into the PAUP 4.0b4 program (32), where phylogenetic trees were generated using a neighbor-joining algorithm.

Nucleotide sequence accession numbers. The four -galactosidase sequences were deposited in the EMBL nucleotide sequence database with the accession numbers AJ272131 (BIF1), AJ224434 (BIF2), AJ224435 (BIF3), and AJ224436 (INF1).

	RESULTS

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Isolation of -galactosidase genes from B. bifidum DSM20215. Genes encoding -galactosidase from B. bifidum DSM20215 were cloned by shotgun cloning. Chromosomal DNA was isolated, cut with restriction enzymes, and inserted into cloning vectors as described in Materials and Methods. Ligation mixtures were transformed into -galactosidase-deficient E. coli cells, and -galactosidase-producing transformants were identified on X-Gal indicator plates. Ligation mixtures with PstI-restricted B. bifidum DSM20215 chromosomal DNA gave rise to five positive blue clones out of approximately 1,500 screened transformants, and mixtures with KpnI-restricted DNA resulted in one positive clone out of approximately 600 transformants. Restriction enzyme analysis indicated that four of the five PstI clones were identical, whereas the fifth PstI clone was different from the four identical clones. The PstI clones were denoted pBIF1 and pBIF3, respectively, and the single KpnI clone was denoted pBIF2. The positions of the -galactosidase genes on the cloned fragments were examined by subcloning the inserts of the plasmids pBIF1, pBIF2, and pBIF3, respectively, as described below.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Map of plasmids pBIF1, pBIF2, pBIF3, and pINF1. Black boxes indicate the cloning vector pBluescript KS(), grey boxes show the position of -galactosidase genes, and white boxes symbolize cloned sequences outside the -galactosidase genes. Restriction enzyme sites used for mapping the -galactosidase genes are shown above the maps, and the sites used as anchors in DNA sequencing by primer walking are indicated by arrows below the maps.

Isolation of -galactosidase genes from B. infantis DSM20088. Genes from B. infantis DSM20088 encoding -galactosidase were isolated as described above for B. bifidum. Chromosomal DNA was restricted with KpnI, inserted in a cloning vector, and transformed into -galactosidase-deficient E. coli cells as described in Materials and Methods. Nine -galactosidase producing clones out of approximately 5,000 transformants were isolated. DNA sequencing showed that all the clones were identical. One of the clones, pINF1, was selected for further analysis. DNA sequencing of a 4.3-kb KpnI fragment by primer walking from the ends of the fragment revealed an open reading frame of 690 amino acid codons corresponding to a molecular mass of 77 kDa (EMBL accession number AJ224436).

Characterization of -galactosidase genes from B. bifidum and B. infantis. A comparison of the open reading frames in plasmids pBIF1, pBIF2, pBIF3, and pINF1 showed only a minor degree of protein sequence similarity between the encoded -galactosidases (Fig. 2). Especially, the pINF1 sequence seemed to be distantly related to the other three genes and to the E. coli lacZ gene, as indicated by the short stretches of homology that the BestFit analysis returned (data not shown). Despite the fact that all four genes are bifidobacterial -galactosidase genes and that three of them were derived from the same strain, they showed surprisingly little resemblance to each other.

View larger version (167K): [in this window] [in a new window]   FIG. 2.   Amino acid sequence comparison of active-site regions in selected -galactosidases. (A) Sequences corresponding to the region around the catalytic Glu461 in the lacZ enzyme of E. coli were aligned using the ClustalX program. The BIF1, BIF2, BIF3, and INF1 sequences were obtained in this study. The other sequences are identified by database accession numbers. The conserved glutamic acid residue (E) is shown in frames, and conserved residues within classes I, II, and III are shaded. (B) Neighbor-joining analysis of the alignment shown in Fig. 2A. The Sulfolobus sequences were used as an outgroup. Results from a bootstrap analysis (n = 100) are shown for the junctions with a value above 80.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Identification of a galactose binding domain in BIF3. The amino acid sequence of the BIF3 -galactosidase (this study) was aligned to (i) galactose binding domains in sialidase from M. viridifaciens (accession no. D01045) and galactose oxidase from D. dendroides (accession no. M86819), (ii) sialidase from C. septicum (accession no. X63266), and (iii) a protein of unknown function from Streptomyces coelicolor (accession no. AL031155). Amino acid residues, which have been found by X-ray crystallography to interact with the bound galactose moiety in sialidase from M. viridifaciens, are marked with an asterisk below the sequences.

View larger version (41K): [in this window] [in a new window]   FIG. 4.   Comparison of UTS immediately upstream of the ATG start codon in Bifidobacterium genes and the 3'-terminal consensus sequence in Bifidobacterium genes encoding 16S rRNA. Potential base pairing to the 3'-terminal rRNA sequence is indicated by underlined nucleotides. Possible sequences facilitating ribosome binding in E. coli are shaded in the BIF1, BIF2, BIF3, and INF1 sequences. The boldface type indicates the ATG start codon. The sequences are identified by database accession numbers.

Heterologous expression of B. bifidum and B. infantis -galactosidases in E. coli. The Bifidobacterium -galactosidase genes on plasmids pBIF1, pBIF2, pBIF3, and pINF1 were expressed in E. coli under growth conditions which would normally repress expression from the inducible E. coli lacZ promoter located in the flanking region in the cloning vector. This observation indicated that endogenous, internal bifidobacterial sequences upstream of the -galactosidase genes may serve as transcription initiation signals in E. coli. Similarly, initiation of translation may be facilitated by the putative E. coli ribosome binding sites (AGGA) (Fig. 4) which were observed immediately upstream of the open reading frame in all the -galactosidase genes. The -galactosidase activity of E. coli cells expressing BIF1, BIF2, BIF3, or INF1 was exclusively found in cell extracts (BIF1, BIF2, and INF1) or in cell extract and membrane fraction (BIF3), whereas no activity was found in the growth medium.

View this table: [in this window] [in a new window]   TABLE 1.   Structural properties of recombinant B. bifidum and B. infantis -galactosidases produced in E. coli

Substrate specificity and transgalactosylation properties. The substrate specificity of the four -galactosidases was examined in enzyme assays with the following chromogenic substrates: ONP--D-galactose, ONP--D-glucose, ONP--D-xylose, ONP--D-fucose, and ONP--D-galactose-6-phosphate. All the -galactosidases predominantly hydrolyzed ONP--D-galactose, and less than 10% of the activity observed with ONP--D-galactose was measured with the other substrates.

View larger version (102K): [in this window] [in a new window]   FIG. 5.   Transgalactosylation with -galactosidases from B. bifidum and B. infantis. Various amounts of cell lysates of recombinant E. coli strains expressing the indicated -galactosidase genes were used to obtain comparable lactose conversion profiles. Elix'or is a commercial galactooligosaccharide product obtained from Borculo Whey Products, Borculo, The Netherlands.

	DISCUSSION

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In this paper, we present three new -galactosidase genes from B. bifidum DSM20215 and one new -galactosidase gene from B. infantis DSM20088. In a previous report, Dumortier et al. (7) described the purification of one of the three -galactosidases that they found in B. bifidum DSM20215, and they characterized the transgalactosylating activity of the enzyme.

We have now extended these studies by isolation, cloning, and sequencing of the three different -galactosidase genes of B. bifidum, followed by heterologous expression in E. coli and characterization of the individual enzymes. Although the molecular structure and the amino acid sequence of the three enzymes differed widely, they were all highly specific for hydrolysis of the -D-galactosidic bond in lactose as judged from assays with ONP substrates. Furthermore, we found that all three -galactosidases were able to catalyze the formation of galactooligosaccharides by transgalactosylation. This result, however, does not correspond to the data presented by Dumortier et al. (7), who found that B. bifidum DSM20215 contains one transgalactosylating and two hydrolysing -galactosidases. The discrepancy might be explained by the use of different assay conditions, e.g., pH and lactose concentration. The BIF3 enzyme presented here probably corresponds to the transgalactosylase characterized by Dumortier et al. (7), since almost identical molecular weights were observed.

The enzymes BIF1 and BIF2, described in this study, both have a multimeric subunit structure which is very similar to other family 2 -galactosidases, e.g., a subunit molecular mass of approximately 115 kDa, no signal sequence, and no carbohydrate binding domain. In contrast, BIF3 is very different from other enzymes classified as family 2 glycosyl hydrolases. This -galactosidase is active as a monomeric 180-kDa molecule and contains a putative signal sequence which is cleaved off when the enzyme is expressed heterologously in E. coli, resulting in transport of the enzyme to the periplasma, either in a free form or bound to the membrane. Generally, attempts to translocate the E. coli lacZ protein to the extracellular space or to the periplasma of E. coli have failed so far, probably because of membrane jamming or periplasmic toxicity (16, 29, 31). However, since the BIF3 -galactosidase had no such deleterious effects on the E. coli cells in this study and since this enzyme is active as a monomer, the BIF3 enzyme might be of potential use as a reporter gene for protein export studies in gram-positive as well as gram-negative bacteria. Another unique feature of the BIF3 -galactosidase is the presence of a putative galactose binding site. Amino acid sequence alignments of the C-terminal part of the BIF3 -galactosidase and the galactose binding domains of sialidase and galactose oxidase showed a high degree of similarity and, notably, a strict conservation of the amino acid residues known to be involved in carbohydrate binding. Whether the BIF3 galactose binding domain is actually involved in substrate binding, as has been suggested for other galactose binding domains (9), is presently not known.

In the present report, we also describe the isolation, cloning, and sequencing of a -galactosidase gene from B. infantis which encoded a polypeptide of 73 kDa (INF1). This -galactosidase was a dimeric, intracellular enzyme specific for -galactosidic linkages and showed transgalactosylase activity. Phylogenetic analysis of the gene sequence revealed that INF1 is a family 42 glycosyl hydrolase and that the subunit molecular mass of INF1 is similar to that of typical family 42 polypeptides (70 to 80 kDa). Recently, Hung and Lee (13) described two -galactosidases from B. infantis with estimated subunit sizes of 110 and 73 kDa, the latter probably corresponding to the gene presented in this paper. The reason why we did not isolate the gene encoding the 110-kDa enzyme described by Hung and Lee (13) might be ascribed to the cloning strategy.

The presence of multiple -galactosidases in bacteria is not uncommon. Other bifidobacterial strains have been shown to harbor multiple -galactosidases (25, 37), and Bacillus circulans has also been shown to contain three -galactosidases (38). But why do bacteria contain multiple -galactosidase genes? Van Laere et al. (37) have shown that the two -galactosidases isolated from B. adolescentis are very different with respect to substrate specificity and regulation of gene expression, one being a lactose-hydrolyzing enzyme induced by lactose, the other being a galactooligosaccharide-hydrolyzing -galactosidase induced by lactose and galactooligosaccharides. In this paper, we show that the three -galactosidases from B. bifidum are structurally different: BIF3 is most probably an extracellular enzyme, whereas the two other -galactosidases from B. bifidum are intracellular. However, a solid explanation for the presence of multiple -galactosidases in individual bacterial species has not yet been presented, and further experiments will be needed in order to unravel the physiological role of differences in enzyme localization, substrate specificity, and gene expression.