Extracellular homopolysaccharides and oligosaccharides from intestinal lactobacilli

Journal of Applied Microbiology 2005, 99, 692–702

doi:10.1111/j.1365-2672.2005.02638.x

Extracellular homopolysaccharides and oligosaccharides from intestinal lactobacilli M. Tieking1, S. Kaditzky1, R. Valcheva2, M. Korakli1, R.F. Vogel1 and M.G. Ga¨nzle1 1

TU Mu¨nchen, Lehrstuhl fu¨r Technische Mikrobiologie, Freising, Germany, and 2Laboratoire de Microbiologie Alimentaire et Industrielle, Enitiaa, Nantes Cedex, France

2004/0919: received 9 August 2004, revised 4 November 2004 and accepted 6 November 2004

ABSTRACT M . T I E K I N G , S . K A D I T Z K Y , R . V A L C H E V A , M . K O R A K L I , R . F . V O G E L A N D M . G . G A¨ N Z L E . 2005.

Aims: To characterize lactobacilli isolated from the intestines of ducks or pigs with respect to the production of extracellular homopolysaccharides (HoPS) and oligosaccharides. Methods and Results: Lactobacillus strains of duck or pig origin were screened for HoPS synthesis and >25% of the isolates produced fructans or glucans from sucrose. Glucan-forming strains were found within the species Lactobacillus reuteri and Lactobacillus animalis and fructan-forming strains were found within Lactobacillus mucosae, Lactobacillus crispatus and Lactobacillus acidophilus. The glucan-forming strains of L. reuteri but not L. animalis produced glucose-oligosaccharides in additon to the respective polymers, and two fructan-forming strains of L. acidophilus produced kestose. Genes coding for glycosyltransferases were detected by PCR and partially characterized by sequence analysis. Conclusions: A large proportion of lactobacilli from intestinal habitats produce HoPS from sucrose and polysaccharide formation is generally associated with the formation of glucose- and fructose oligosaccharides. Significance and Impact of the Study: The characterization of the metabolic potential of intestinal lactobacilli contributes to the understanding of the molecular basis of autochthony in intestinal habitats. Moreover, this is the first report of glucose-oligosaccharide production during growth of lactobacilli, and one novel fructosyltransferase and one novel glucansucrase were partially characterized on the genetic level. Keywords: extracellular polysaccharides, fructan, fructose-oligosaccharides, glucan, glucose-oligosaccharides, intestinal lactobacilli, Lactobacillus reuteri.

INTRODUCTION Several species of lactobacilli are autochthonous to the gut of man and animals (Hammes and Hertel 2003; Tannock 2004). The molecular basis of autochthony remains a fascinating challenge to microbiologists and Lactobacillus reuteri has been frequently used as an example to investigate bacterial metabolic properties essential for persistence in the gut (Tannock 2004). Several genes were identified in L. reuteri, a xylose isomerase and Correspondence to: M.G. Ga¨nzle, University of Alberta, Dept. of Agricultural, Food, and Nutritional Science, 4/10 Agriculture/Forestry Centre, Edmonton, AB, Canada T6G 2P5 (e-mail: [email protected]).

methionine sulfoxide reductase, that are specifically expressed upon colonization of the rodent gut (Walter et al. 2003). Moreover, strains of L. reuteri are characterized by an exceptional metabolic diversity that may contribute to their persistence in intestinal ecosystems. Several strains of L. reuteri produce reuterin or reutericyclin, antibacterial compounds that are unique among the lactic acid bacteria (Rodriguez et al. 2003; Ga¨nzle 2004). In recent years, it was observed that L. reuteri strains produce extracellular homopolysaccharides (HoPS) of the fructan- and glucan-type (van Geel-Schutten et al. 1998; Tieking et al. 2003). A large structural variety of glucans from L. reuteri has been reported (Kralj et al. ª 2005 The Society for Applied Microbiology

EPS AND OLIGOSACCHARIDE PRODUCTION BY INTESTINAL LACTOBACILLI

2003) and the fructan polymers inulin and levan are produced by L. reuteri enzymes (van Hijum et al. 2002 and 2004). The formation of extracellular glucans and fructans from sucrose is catalysed by enzymes termed glucosyltransferases (Gtf) or fructosyltransferases (Ftf) respectively. In the past, a variety of gtf and ftf genes have been characterized from Leuconostoc and Streptococcus strains (Monchois et al. 1998; Monsan et al. 2001) and more recently from L. reuteri (van Hijum et al. 2002 and 2004; Kralj et al. 2003). These enzymes are generally cell-wall anchored or secreted into the environment (Monsan et al. 2001). Gtfs and Ftfs use sucrose as substrate and catalyse the hydrolysis of sucrose, the formation of glucose- or fructose-oligosaccharides and the formation of glucan- or fructan-polymers respectively (Monsan et al. 2001). Whereas, the formation of the fructose-oligosaccharide (FOS) kestose by lactobacilli has been described (Korakli et al. 2003), the formation of glucose-oligosaccharides (GOS) during growth of lactobacilli has to date not been observed. Whereas several intestinal lactobacilli are known to produce HoPS (Duboc and Pridmore 2003; Kralj et al. 2003; Tieking et al. 2003), it remains unknown whether or not HoPS formation is a general property of intestinal lactobacilli. The objective of this study therefore was to determine the proportion of HoPS forming lactobacilli among isolates of intestinal origin, and to investigate the ability of these strains to produce FOS or GOS from sucrose. Therefore, lactobacilli isolated from two different habitats, the intestine of ducks and pigs, were screened for the production of glucans and fructans. This screening was based on the detection of extracellular HoPS and oligosaccharides, and the detection of gtf- and ftf-genes with PCR methods.

MATERIALS AND METHODS Strains, media and growth conditions A total of 42 lactobacilli were screened for HoPS-production and oligosaccharide-synthesis. Twenty three strains were previously isolated from the crop or intestinum of ducks and identified to species level (Kurzak et al. 1998). The selection of bacteria comprised strains of Lactobacillus salivarius ssp. salicimus (5), Lactobacillus agilis (1), Lactobacillus delbrueckii ssp. lactis (1), L. reuteri (10), Lactobacillus animalis (2) and Lactobacillus acidophilus (4). To avoid selection of isogenic organisms, strains of the same species were chosen from different randomly amplified polymorphic DNA (RAPD) clusters. Furthermore, a total of 19 lactobacilli were isolated from the faeces of 20 pigs, characterized to species level with amplified fragment length polymorphism (AFLP) and partial sequences of the 16S rDNA, and screened for HoPS production as described below.

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All strains were grown anaerobically at 37
C in MRSmedium modified according to Stolz et al. (1995), containing 5 g l)1 fructose, 5 g l)1 glucose and 10 g l)1 maltose (mMRS). For the HoPS screening, cells were grown on mMRS-sucrose containing 120 g l)1 sucrose as the sole carbon source. Where appropriate, 15 g l)1 agar was added for solid media. The pH was adjusted to 6Æ2 before autoclaving and sugars were autoclaved separately. Lactobacillus sanfranciscensis 1Æ392 and L. reuteri TMW 1Æ106 were used as positive controls for the formation of fructan and glucan (Tieking et al. 2003). Isolation of lactobacilli from pig faeces For the determination of lactobacilli in the intestinal microflora of pigs, pig faeces was diluted and plated on mMRS. Colonies with different morphologies were subcultured twice by dilution plating on mMRS plates to obtain pure cultures. As a subsequent step, DNA was isolated from the different isolates and AFLP was performed as described below. At least one representative strain of each AFLP cluster was chosen and 16S rDNA was partially amplified using the primer combination 616V and 609R (Table 1). Identification to species level was achieved by sequencing the 16S rDNA amplicons. General molecular techniques and PCR General techniques regarding DNA manipulations and agarose gel electrophoreses were performed as described by Sambrook et al. (1989). Chromosomal DNA was isolated according to the method of Lewington et al. (1987). PCR was carried out in thermocyclers (Eppendorf, Hamburg, Germany) by using taq polymerase from Promega (Mannheim, Germany) and dNTPs from Diagonal (Waldeck, Germany). In general, 3 mmol l)1 MgCl2, 0Æ4 mmol l)1 dNTPs, 0Æ5 lmol l)1 primer and 1Æ5 U taq were used per reaction; for AFLP-PCRs 1Æ5 mmol l)1 MgCl2, )1 0Æ2 mmol l dNTPs, 0Æ4 U taq, 0Æ3 lmol l)1 primer (PreM, Pre-E and M-CT) and 0Æ05 lmol l)1 primer E*-A were used respectively. The PCR-products were purified using the QIAquick PCR Purification Kit (Quiagen, Hilden, Germany) according to the instructions of the supplier and sequenced by SequiServe (Vaterstetten, Germany). Nucleotide and amino acid (aa) sequence analysis was carried out using the DNASis for Windows software (Hitachi Software Engineering Co, Yokohama, Japan). Amplified fragment length polymorphism The AFLP typing was performed essentially as described by Vos et al. (1995) as modified by Schmidt et al. (2003) using the restriction enzymes MseI and EcoRI. For clustering of

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Primer name and sequence (5¢–3¢) Primers dexwobV dexwobR DexreuV DexreuR levV levR Dexani_exV Dexani_exR 616V 609R

GAYGCIGTIGAYAAYGTI YTGRAARTTISWRAAICC GTGAAGGTAACTATGTTG ATCCGCATTAAAGAATGG GAYGTITGGGAYWSITGGC TCITYYTCRTCISWIRMCAT GCTGCCTACAAAGTTGGC ACCCCATACCTTGAGTCC AGAGTTTGATYMTGGCTCAG

Conserved aa sequence*

DAVDNV EGFSNF

GATGAGTCCTGAGTAAC GACTGCGTACCAATTCA GACTGCGTACCAATTCA GATGAGTCCTGAGTAACT

gtf-screening gtf-screening gtf-screening gtf-screening ftf-screening ftf-screening RT-PCR RT-PCR Species identification Species identification AFLP AFLP AFLP AFLP

GACGATGAGTCCTGAG TACTCAGGACTCAT CTCGTAGACTGCGTACC AATTGGTACGCAGTCTAC

AFLP AFLP AFLP AFLP

DVWDSWP DEV(I;L)ER

ACTACYVGGGTATCTAAKCC

Pre-M Pre-E E*-A M-CT AFLP-adapter M1 M2 E1 E2

Purpose

Table 1 Primers used for species identification and screening for HoPS biosynthetic enzymes

Primers used for PCR with I for Inosin; Y for C or T; S for G or C; W for A or T; R for A or G; M for A or C according to International Union of Biochemistry group codes. Primers used for AFLP were those described by Vos et al. (1995). Selective base sides of AFLP primers are underlined. AFLP oligonucleotides termed M are appendant to MseI, those termed E to EcoRV restriction sites, the primer E*-A was fluorescence labelled with Cy5 to allow laser detection of the amplicons. *Conserved aa sequences within Gtf and Ftfs are indicated that were used for the construction of degenerated primers.

strains, TIFF images of the gels were analysed with the BioNumerics software package (Applied Maths, Sint-Martens-Latem, Belgium). The Pearson correlation was used for the curve based pair wise similarity calculation and the dendrogram was calculated using the UPGMA algorithm. Sucrose metabolism, screening for HoPS synthesis and determination of the monosaccharide composition Screening for HoPS production was performed essentially as previously described (Tieking et al. 2003). In brief, high molecular weight (HMW) extracellular polysaccharides (EPS) were detected with a Superdex 200 gel permeation chromatography (GPC) column (Amersham Pharmacia Biotech, Uppsala, Sweden), coupled to a refractive index (RI) detector (Gynkotek, Germering, Germany). The fermentation medium did not contain compounds with a relative molecular weight >105 (data not given). To determine the monosaccharide-composition of the EPS,

EPS from the supernatants was precipitated with two volumes of cold ethanol, vacuum dried, redissolved and dialysed against water (molecular weight cut-off 12– 14 kDa). Afterwards, the EPS was hydrolysed by the addition of perchloric acid to a concentration of 5% to hydrolyse fructans or H2SO4 to a concentration of 2 mol l)1 to hydrolyse glucans and incubated for 2 h at 80
C. Acid hydrolysates were neutralized with 2 mol l)1 KOH and the sugar composition of the samples was analysed by HPLC using a polyspher CHPB column (Merck, Darmstadt, Germany) and water at a flow rate of 0Æ4 ml min)1 as previously described (Tieking et al. 2003). In order to calculate carbon recoveries for the sucrose metabolism of selected HoPS-positive strains, the concentrations of mono- and disaccharides as well as the metabolites lactate, acetate, ethanol and mannitol in culture supernatants of L. reuteri TMW 1Æ106, TMW 1Æ974 and 1Æ976 were quantified by HPLC as previously described (Korakli et al. 2003). Lactate, acetate and ethanol were quantified using a polyspher OAKC column eluted with

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5 mmol l)1 H2SO4 at a flow of 0Æ4 ml min)1. Glucose, fructose, sucrose, maltose and mannitol were quantified using a polyspher CHPB column eluted with water at a flow of 0Æ4 ml min)1. In order to quantify glucose moieties recovered in HMW glucans from L. reuteri TMW 1Æ106 and TMW 1Æ974, the glucans from these strains were purified by ethanol precipitation and dialysis, and dried by lyophilization as described above. These purified glucans were redissolved in water to 1, 2, 5 and 10 g l)1 and analysed by GPC. The same glucan stock solutions were hydrolysed as described above and the glucose concentrations in the hydrolysates were determined. Screening for oligosaccharide production and preliminary characterization of novel oligosaccharides Cell-free culture supernatants were analysed for oligosaccharides by high-performance anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD) as previously described by Thiele et al. (2002). No oligosaccharide peaks were present in unfermented mMRS (data not given). In order to characterize GOS from L. reuteri TMW 1Æ106, this strain was grown on mMRS-sucrose, cells were removed by centrifugation, and 22Æ5 ml of the culture supernatant was lyophilized and redissolved in 2 ml of deionized water. The concentrated supernatant was injected on a Superdex 75 Prepgrade GPC column (Amersham Pharmacia Biotech) with deionized water as mobile phase at a flow rate of 4Æ0 ml min)1, and collected in 72 fractions of 5 ml. The GOS in these fractions were detected with HPAEC-PAD, and fractions with GOS were pooled, lyophilized, redissolved in 0Æ6 ml deionized water and separated on a Superdex Peptide GPC column (Amersham Pharmacia Biotech) with deionized water as mobile phase at a flow rate of 0Æ4 ml min)1. Carbohydrates with a degree of polymerization (DP) from n ¼ 1 to n ¼ 4 (fructose, maltose, raffinose and stachyose) were used to calibrate the Superdex Peptide GPC column. Fractions of 1 ml were collected and analysed for the presence of GOS. The fractions containing purified GOS were lyophilized, redissolved in 0Æ5 ml of 4 mol l)1 hydrochloric acid and incubated at 90
C for 2 h. Hydrochloric acid was removed by drying the sample at 70
C with a constant nitrogen flow, the residue was redissolved in 0Æ5 ml deionized water and the monosaccharides in the hydrolysate were analysed by HPAEC-PAD. Screening for glucosyl- and fructosyltransferases genes For the detection of glucosyltransferase genes in lactobacilli, primers were constructed (Table 1) targeting highly conserved regions in the catalytic domains of known bacterial

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glucansucrases (Monchois et al. 1998). The primer pair LevV and LevR was used to amplify fragments of fructosyltransferase genes (Tieking et al. 2003). Detection of glucansucrase gene-expression Total RNA was isolated from L. animalis TMW 1Æ971 grown to the exponential growth phase (OD 578 nm ¼ 0Æ2) in mMRS-sucrose. A 0Æ6 ml was taken from the culture and resuspended in 1Æ2 ml stop buffer (RNA protect bacteria reagent; Quiagen). The isolation of RNA was performed using the RNeasy Plant Mini Kit (Quiagen) according to the instructions of the manufacturer. In the RNA preparations, DNA was digested by incubation with RQ1 RNAse free DNAse (Promega). RT-PCR was performed using 100 U M-MLV reverse transcriptase and primed with 20 lg ml)1 of hexameric random primers (rRNase H minus and random primers from Promega). From the cDNA library, internal fragments of the glucansucrase genes were amplified using the primer pair dexani_exV/R for the gtf gene of L. animalis 1Æ971 (Table 1). The PCR was also carried out with DNAse digested RNA preparations to ensure complete hydrolysis of chromosomal DNA in the RNA preparations. Nucleotide accession number The nucleotide sequences of the L. acidophilus TMW 1Æ989 levansucrase and the L. animalis TMW 1Æ971 glucansucrase genes have been assigned the EMBL accession numbers AJ698830 and AJ698831 respectively.

RESULTS Identification of lactobacilli from the pig faeces and their ability for HoPS formation In the TMW strain collection, the lactobacilli previously isolated from ducks are deposited (Kurzak et al. 1998). In order to evaluate the potential for HoPS production of lactobacilli from a second well-characterized intestinal habitat, lactobacilli were isolated from pig faeces. A total of 19 pig isolates and nine reference strain were clustered based on their AFLP patterns using the BioNumerics software package (Fig. 1). At least one representative strain of each cluster was chosen and identified to species level on the basis of partial 16S rDNA sequences. With the exception of two strains of L. mucosae, all strains exhibiting a similarity level of 55% or higher could be allotted to the same species. The total Lactobacillus cell count was 5 · 108– 2 · 109 CFU g)1 and the species L. mucosae (two strains), L. reuteri (6), Lactobacillus crispatus (7), L. acidophilus (1) and Lactobacillus johnsonii (1) were identified. This composition of lactobacilli on species level is in general agreement with

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100

80

60

40

20

696 M . T I E K I N G ET AL.

Lactobacillus ruminis

TMW 1·804

Lactobacillus agilis

TMW 1·803

Lactobacillus alimentarius

TMW 1·11

Lactobacillus mucosae

TMW 1·145 a, b

Lactobacillus reuteri

TMW 1·272 a

Lactobacillus reuteri

TMW 1·279 a

Lactobacillus reuteri

TMW 1·285 a

Lactobacillus reuteri

TMW 1·146 a, b

Lactobacillus reuteri

TMW 1·194 a

Lactobacillus reuteri

TMW 1·138 a

Lactobacillus crispatus

TMW 1·143 a, b

Lactobacillus crispatus

TMW 1·209 a

Lactobacillus crispatus

TMW 1·267 a

Lactobacillus crispatus

TMW 1·195 a

Lactobacillus crispatus

TMW 1·144 a, b

Lactobacillus crispatus

TMW 1·193 a

Lactobacillus crispatus

TMW 1·139 a, b

Lactobacillus amylovorus

TMW 1·694

Lactobacillus vaginalis

TMW 1·1144

Lactobacillus spec.

TMW 1·288 a

Streptococcus alactolyticus

TMW 1·140 a, b

Lactobacillus acidophilus

TMW 1·142 a, b

Lactobacillus salivarius

TMW 1·810

Lactobacillus mucosae

TMW 1·141 a, b

Lactobacillus mucosae

TMW 1·81

Lactobacillus johnsonii

TMW 1·1179

Lactobacillus johnsonii

TMW 1·265 a

Lactobacillus animalis

TMW 1·806

Fig. 1 AFLP cluster analysis of 19 lactobacilli isolated from pig faeces and nine reference strains. The dendrogram was calculated based on TIFF images of AFLP gels and UPGMA (unweighted pair group method with arithmetic averages) cluster analysis of the bands. (a) denotes pig isolates, other strains are reference strains previously identified to species level and (b) denotes isolates identified based on partial sequences of their 16S rRNA genes

previous investigations (Leser et al. 2002; Hammes and Hertel 2003) although Lactobacillus amylovorus and L. salivarius were not found. Screening of lactobacilli isolated for HoPS synthesis Lactobacilli of intestinal origin were inoculated in mMRSsucrose medium and culture supernatants were analysed for polysaccharides. Eight strains of the 23 lactobacilli of duck origin and five strains of the 19 lactobacilli of pig origin were identified as HoPS-producers (Table 2). All polymers had a relative molecular weight equal or larger than the exclusion limit of the GPC column, 5 · 106. The monomer composition

of the EPS was determined by hydrolysis of the purified polymers and HPLC-analysis of the monosaccharides. Of the duck isolates, three strains of L. reuteri and one strain of L. animalis were identified as glucan producers, whereas four strains of L. acidophilus produced fructan (Table 2). Of the pig isolates, two strains of L. reuteri produced glucans and three fructan-forming strains of the species L. crispatus, L. acidophilus and L. mucosae were identified. Production of oligosaccharides by HoPS-producing strains To evaluate whether oligosaccharides are formed by HoPS-positive strains, culture supernatants of the fructan

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Table 2 HoPS producing lactobacilli isolated from ducks or pigs

Organism Lactobacilli of duck origin L. reuteri TMW 1Æ974 L. reuteri TMW 1Æ976 L. reuteri TMW 1Æ979 L. animalis TMW 1Æ971 L. acidophilusi TMW 1Æ986 L. acidophilus TMW 1Æ987 L. acidophilus TMW 1Æ989 L. acidophilus TMW 1Æ991 Lactobacilli of porcine origin L. reuteri TMW 1Æ272 L. reuteri TMW 1Æ138 L. crispatus TMW 1Æ144 L. acidophilus TMW 1Æ142 L. mucosae TMW 1Æ141

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EPS formation

Oligosaccharide formation

Glycosyltransferase genes detected with PCR

Glucan Glucan Glucan Glucan Fructan Fructan Fructan Fructan

GOS GOS GOS – FOS ND FOS ND

ftf and gtf ftf and gtf ftf and gtf gtf ftf ftf ftf ftf

Glucan Glucan Fructan Fructan Fructan

GOS GOS ND ND ND

None None ftf ftf ftf

ND, not determined; ftf, fructosyltransferase gene; gtf, glucosyltransferase gene.

producing strains L. acidophilus TMW1Æ986 and 1Æ989 and of all glucan producing strains were analysed by HPAEC-PAD. Strains TMW1Æ986 and 1Æ989 produced kestose on mMRS-sucrose as identified by external standards of kestose and nystose (Fig. 2 and data not given). All glucan-positive strains with the exception of L. animalis produced oligosaccharides (Figs 2 and 3). The retention time of all oligosaccharide peaks from L. reuteri strains were identical to the retention time of oligosaccharides formed by L. reuteri TMW 1Æ106 (Fig. 3). This strain was used in this study as a positive control for glucan formation because of a well characterized sucrose metabolism (Tieking et al. 2003 and unpublished results). By the use of external standards, it

could be excluded that the peaks detected in L. reuteri supernatants are attributable to leucrose, panose, maltose or maltotriose (Fig. 2 and data not given). The oligosaccharide from L. reuteri TMW 1Æ106 could be purified to homogeneity as judged by HPAEC-PAD in two consecutive preparative GPC runs (Fig. 3). Glucose was the major carbohydrate (>95% of peak area) that was found in the acid hydrolysate of the oligosaccharide, demonstrating that it is a GOS. The retention time of the GOS on the Superdex peptide GPC column demonstrates that its DP is >4. By extrapolation of the calibration based on DP1–DP4 carbohydrates, it can be estimated to have a DP of 10. The comparison of the retention time of the GOS on the

500

300

kestose

sucrose

fructose

glucose

TMW 1·986 oligosaccharide

oligosaccharide

sucrose

fructose

400

glucose

mannitol

Detector signal (mv)

TMW 1·974

200 0

10

20

30 0 Retention time (min)

10

20

30

40

Fig. 2 HPLC analysis of culture supernatants from Lactobacillus acidophilus TMW1Æ986 and Lactobacillus reuteri TMW 1Æ974 grown on mMRSsucrose for 48 h. Peaks were assigned based on external standards. Kestose formation was also observed in Lactobacillus acidophilus TMW 1Æ989 (data not given). Mannitol, glucose, fructose, sucrose and kestose were identified based on external standards; for characterization of the oligosaccharide peak at 36 min see (Fig. 3) and text ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 692–702, doi:10.1111/j.1365-2672.2005.02638.x

698 M . T I E K I N G ET AL.

210

(a)

(b) 400

G7 G6

Detector signal (mV)

1

G5

G9

320

G10 G4

2

190

240

3 4

Detector response (mV)

G8

200

180 5 160 6 170 36

39

42

34

36

38

40

42

Retention time (min)

Fig. 3 (a) GOS-peaks from culture supernatants Lactobacillus reuteri strains TMW 1Æ138 (1), TMW 1Æ272 (2), TMW 1Æ979 (3), TMW 1Æ976 (4), 1Æ974 (5) and 1Æ106 (6) obtained on HPAEC-PAD. The identical retention time indicate identical oligosaccharides. (b) Separation of the GOS purified from L. reuteri TMW 1Æ106 by two consecutive GPC runs (lower trace) and a standard mixture of maltose-oligosaccharides (DP4–DP10, labelled as G4 through G10, upper trace)

HPAEC column with the retention time of maltose oligosaccharides (DP4–DP10) further substantiates that the GOS has a DP of about 10 (Fig. 3). To estimate the amounts GOS formed by L. reuteri TMW 1Æ106, TMW 1Æ974 and TMW 1Æ976, carbon recoveries from sucrose were calculated. Generally, the HPLC analysis in our hands allows to establish carbon recoveries in the range of 90–110% after growth of heterofermentative lactobacilli in mMRS with various carbon sources (Korakli et al. 2003 and data not given). The metabolites glucan, glucose, fructose, mannitol, glucan, lactate, acetate and ethanol were directly quantified by appropriate HPLC methods. The amount of glucose in GOS was derived indirectly from the molar difference of the sucrose that was consumed and the metabolites that were produced from glucose. For example, in fermentations with 120 g l)1 sucrose and 5 g l)1 fructose as carbon sources, L. reuteri TMW 1Æ974 consumed 66 mmol l)1 sucrose. Fructose released from sucrose accumulated in the medium or was recovered as mannitol. Of the glucose moieties in sucrose, 30 mmol l)1 (45%) were recovered in the metabolites lactate, acetate and ethanol and 27 mmol l)1 (40%) of the glucose was recovered as high-molecular weight glucan. Thus, glucose moieties in the GOS account for c. 9 mmol l)1 or 14% of the glucose moities in sucrose. A comparable ratio of sucrose hydrolysis to glucan- and GOS formation was found in the strains L. reuteri TMW 1Æ976 and TMW 1Æ106 (data not given).

Molecular screening for genes encoding glucosyland fructosyltransferases A screening for ftf and gtf genes in the HoPS producing strains was performed with degenerated primers, that were obtained from conserved sequences of known bacterial enzymes. Chromosomal DNA of the fructan- or glucanforming lactobacilli was used as template. Using the specific primers dexreuV/dexreuR targeting the gtfA nucleotide sequence of L. reuteri LB 121 (Kralj et al. 2002), The PCRproducts with the expected size of 600 bp were obtained from all glucan-forming L. reuteri strains of duck origin (Table 2). Sequencing revealed four identical sequences with 90% identity to the internal aa fragment of the GtfA enzyme. No gtf-PCR product was obtained with the chromosomal DNA of the glucan positive L. reuteri strains from pigs, neither with dexwobV/R, nor with primers targeting the gtfA gene (Table 2). Using the primer combination DexwobV/DexwobR, a PCR product was obtained with the DNA from L. animalis TMW1Æ971. Sequencing of the 1200-bp product and analysis of the deduced aa-sequence revealed high homology to bacterial Gtfs; the enzyme belongs to the family 70 of glycosyl hydrolases (http://pfam.wustl.edu). Blast searches revealed the highest degree of identical aa (57%) with a glucansucrase from Leuconostoc mesenteroides (accession number U81374). Alignments of the internal Gtf fragment

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of L. animalis TMW1Æ971 and the four most similar Gtf sequences available in public databases are given in Fig. 4. The fragment comprises the catalytical core of the enzyme and harbours aa residues that are putatively involved in catalysis, binding of acceptor molecules and stabilizing the transition state respectively (Monchois et al. 1998). The expression of the gtf gene of L. animalis TMW 1Æ971 was shown by using PCR targeting an internal sequence using cDNA as template (data not given). Using the primer combination LevV/R, PCR products of 800-bp length were obtained with the fructan-forming organisms L. acidophilus TMW 1Æ144, TMW 1Æ986, TMW 1Æ987, TMW 1Æ989 and TMW 1Æ991. Ftf-amplicons were

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furthermore obtained with the DNA of the fructan-forming strains of L. mucosae and L. crispatus (Table 2). The ftfamplicons obtained from four L. acodipholus strains were sequenced and the amplification products showed 100% identity of the four gene fragments (data not given). Analyses of the deduced aa sequence revealed high homology to bacterial ftfs; the enzymes belong to the family 68 of the glycosyl hydrolases. The highest degree of identical aa (68%) was observed with a levansucrase from L. reuteri (AF465251). An alignment with the five most similar sequences is given in Fig. 5. Remarkably, ftf-amplicons were obtained also from two strains of L. reuteri that did not produce fructans.

Fig. 4 Alignment of the internal Gtf fragment of Lactobacillus animalis 1Æ971 with the four most similar sequences from Leuconostoc mesenteroides NRRL B-512F (LeuconDsrT, AB020020), Lc. mesenteroides B-742CB (LmesDsrb742, AF294469), Lc. mesenteroides NRRL B-512F (LeumesDEX, U81374) and Lactobacillus reuteri LB 121 (Lbreuteri, AX306822). Regions with strong homologies among bacterial Gtfs (Monchois et al. 1998) are shaded grey. () Putative catalytical residue; (() putative residue that may play a role in the binding with acceptor molecules and in the transfer of the glucosyl residue; ( ) putative residue stabilizing the transition state ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 692–702, doi:10.1111/j.1365-2672.2005.02638.x

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Fig. 5 Alignment of the internal Ftf fragment of Lactobacillus acidophilus with the inulosucrase of Lactobacillus reuteri LB 121 (Inuloreu, AF45943) and the Ftfs of L. reuteri LB 121 (Lbreuteri, AF465251), Lactobacillus johnsonii (Ljohnsoni, AE017202), Streptococcus salivarius (Strepsali, L08445). Regions with strong homologies among Ftfs (Naumoff 2001) are shaded grey; () Amino acid residues that may play a role in catalysis (Meng and Fu¨tterer 2003)

DISCUSSION In this work, Lactobacillus strains isolated from the intestine of ducks and pigs were screened on the biochemical and genetic level for their ability to produce HoPS as well as oligosaccharides. An exceptional high proportion of HoPSforming strains, 30 and 26% of the isolates from ducks and pigs, respectively, was found among intestinal lactobacilli. Furthermore, two novel genes coding for fructosyl- and glucosyltransferases, and a novel glucose-oligosaccharide were identified.

Lactobacillus salivarius, L. reuteri and L. acidophilus were the major components of the duck intestinal lactobacilli (Kurzak et al. 1998) and L. reuteri and L. crispatus were the predominant species in the pig faecal samples. Especially among isolates of L. reuteri, a high proportion of HoPSpositive isolates was found. Because the strains that were selected for this study exhibited different RAPD/AFLP patterns, it can be excluded that two clones of the same strain were chosen for the screening. Some species of lactobacilli were previously found to have a significant potential to produce various HoPS (van Geel-Schutten et al.

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1998; Tieking et al. 2003). Tieking et al. (2003) screened a total of 106 Lactobacillus strains from sourdough for HoPS production and found a high proportion of HoPS-producing strains especially among those species that can also be frequently found in gastrointestinal ecosystems, for example, L. reuteri. Currently, partial or complete sequences of seven gtf genes from L. reuteri are available (Kralj et al. 2003). The PCR based screening method are efficient tools to identify HoPS-producing lactobacilli (Kralj et al. 2003; Tieking et al. 2003). In this study, ftf and gtf genes could be detected by PCR in eight of 10 HoPS producing strains. Two glucan forming strains were PCR-negative. Thus, a screening based on PCR techniques only may lead to false negative results, either because of an inadequate primer design or because target sequences are less conserved than the corresponding sequences in known bacterial glycosyltransferases. Silent genes encoding Ftfs (van Hijum et al. 2002) may result in false positive results. In this study, two ftf amplicons were obtained from L. reuteri strains that did not produce fructans. Two novel glycosyltransferase genes could be detected in our work based on partial aa sequences of their catalytic domains, one Ftf from L. acidophilus strains and one Gtf from L. animalis TMW 1Æ971. In L. animalis, it was verified that the gtf gene is expressed and therefore contributes to the glucan formation observed in this organisms. The types of polysaccharides produced by L. animalis TMW 1Æ971 and L. acidophilus strains remain subject of further studies. In addition to the hydrolysis of sucrose and polymer synthesis, Ftfs and Gtfs from streptococci and Leuconostoc spp. catalyse the synthesis of oligosaccharides from sucrose (Monsan et al. 2001). In this study, all tested HoPS-positive lactobacilli were also able to produce oligosaccharides during growth on mMRS-sucrose. L. acidophilus strains produced the prebiotic trisaccharide kestose, and L. reuteri strains produced a novel GOS with a DP of about 10. The purified enzyme reuteransucrase GtfA from L. reuteri 121, that exhibits high homology to the Gtf-enzymes of L. reuteri strains described in this study, was recently shown to synthesize GOS (Kralj et al. 2004). Panose and maltotriose or isopanose and isomaltotetraose were formed in an acceptor-reaction with maltose or isomaltose respectively. Synthesis of these carbohydrates was not observed in this study because maltose was not present in mMRS-sucrose. The ecological significance of HoPS formation by intestinal lactobacilli remains to be elucidated in further studies. Literature data suggests two possible roles of HoPS in intestinal ecology. (i) Dextran formation improves the starvation survival of Lc. mesenteroides at extremes of pH (Kim et al. 2000) and plant fructans are known to protect biological membranes under stress conditions (Oliver et al. 2001). In analogy, HoPS may enhance the survival of

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lactobacilli during gastro-intestinal transit. (ii) Formation of glucans and fructans by oral streptococci is involved in biofilm formation and adhesion to the tooth enamel (Cvitkovitch et al. 2003). L. reuteri strains that persist in the rodent gut have the ability to form a biofilm on the forstomach epithelia of mice (Tannock 2004) and HoPS production by lactobacilli in vivo may be involved biofilm formation. The large number of HoPS positive strains among intestinal lactobacilli makes these organisms an excellent source of strains or enzymes for the industrial production of poly- and oligosaccharides with a wide range of different glycosidic bonds and various branching patterns. HoPS of the levan- and inulintype, as well as kestose and nystose are known to have prebiotic properties (Dal Bello et al. 2001; Simmering and Blaut 2001; Korakli et al. 2002). Because L. animalis, L. reuteri and L. acidophilus are used as starter cultures for food fermentation and/or probiotics, the ability of these organisms to produce extracellular oligosaccharides and prebiotic polymers creates interesting possibilities to produce fermented food of food additives that possess synbiotic properties, and opens new perspectives in the application of pro-, pre- and synbiotics. REFERENCES Cvitkovitch, D.G., Li, Y.H. and Ellen, R.P. (2003) Quorum sensing and biofilm formation in streptococcal infections. J Clin Invest 112, 1626–1632. Dal Bello, F., Walter, J., Hertel, C. and Hammes, W.P. (2001) In vitro study of prebiotic properties of levan-type exopolysaccharides from lactobacilli and non-digestible carbohydrates using denaturing gradient gel electrophoresis. Syst Appl Microbiol 24, 1–6. Duboc, P. and Pridmore, R.-D. (2003) Levansucrase of Lactobacillus johnsonii. European Patent Application EP 1 357 180 A1. Ga¨nzle, M.G. (2004) Reutericyclin: biological activity, mode of action, and potential applications. Applied Microbiology and Biotechnology 64, 326–332. van Geel-Schutten, G.H., Flesch, F., ten Brink, B., Smith, M.R. and Dijkhuizen, L. (1998) Screening and characterization of Lactobacillus strains producing large amounts of exopolysaccharides. Applied Microbiology and Biotechnology 50, 697–703. Hammes, W.P. and Hertel, C. (2003) The genera Lactobacillus and Carnobacterium. In The Prokaryotes: An Evolving Electronic Resource for the Microbiological Community, 3rd edn. ed. Dworkin, M. http://link.springer-ny.com/link/service/books/10125/. van Hijum, S.A.F.T., van Geel-Schutten, G.H., Rahaoui, H., van der Maarel, M.J.E.C. and Dijkhuizen, L. (2002) Characterization of a novel fructosyltransferase from Lactobacillus reuteri that synthesizes high-molecular-weight inulin and inulin oligosaccharides. Appl Environ Microbiol 68, 4390–4398. van Hijum, S.A.F.T., Szalowska, E., van der Maarel, M.J.E.C. and Dijkhuizen, L. (2004) Biochemical and molecular characterization of a levansucrase from Lactobacillus reuteri. Microbiology 150, 621–630.

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