Comparative analysis of the exopolysaccharide biosynthesis gene clusters from four strains of Lactobacillus rhamnosus

B. Péant1,5, G. LaPointe1,2, C. Gilbert3, D. Atlan3, P. Ward4 and D. Roy1,2

1 STELA Dairy Research Centre, Room 1316, Pavillon Paul-Comtois, Université Laval, Quebec, QC, Canada G1K 7P4
2 Nutraceuticals and Functional Foods Institute (INAF), Université Laval, Quebec, QC, Canada G1K 7P4
3 Laboratoire de Microbiologie et de Génétique Moléculaire, UMR CNRS/UCBL/INSA 5122, Université Lyon 1, 69622, Villeurbanne, France
4 Food Research and Development Centre, Agriculture and Agri-Food Canada, St Hyacinthe, Quebec, Canada J2S 8E3
5 Hopital Notre-Dame, 560 Sherbrooke Est, Montréal, QC, Canada H2L 4M1

Correspondence
Gisèle LaPointe
gisele.lapointe{at}fsaa.ulaval.ca


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The exopolysaccharide (EPS) biosynthesis gene clusters of four Lactobacillus rhamnosus strains consist of chromosomal DNA regions of 18·5 kb encoding 17 ORFs that are highly similar among the strains. However, under identical conditions, EPS production varies considerably among these strains, from 61 to 1611 mg l–1. Fifteen genes are co-transcribed starting from the first promoter upstream of wzd. Nevertheless, five transcription start sites were identified by 5'-RACE PCR analysis, and these were associated with promoter sequences upstream of wzd, rmlA, welE, wzr and wzb. Six potential glycosyltransferase genes were identified that account for the assembly of the heptasaccharide repeat unit containing an unusually high proportion of rhamnose. Four genes involved in the biosynthesis of the sugar nucleotide precursor dTDP-L-rhamnose were identified in the EPS biosynthesis locus, which is unusual for lactic acid bacteria. These four genes are expressed from their own promoter (P2), as well as co-transcribed with the upstream EPS genes, resulting in coordinated production of the rhamnose precursor with the enzymes involved in EPS biosynthesis. This is believed to be the first report demonstrating that the sequence, original organization and transcription of genes encoding EPS production are highly similar among four strains of Lb. rhamnosus, and do not vary with the amount of EPS produced.


Abbreviations: BPGN, bacterial polysaccharide gene nomenclature; CPS, capsular polysaccharide; EPS, exopolysaccharide; LAB, lactic acid bacteria; RACE, rapid amplification of cDNA ends; UP element, upstream enhancer element

The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY659976 (ATCC 9595), AY659977 (RW-6541M), AY659978 (strain R) and AY659979 (RW-9595M).


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Interactions between bacteria and human host cells are mediated by cell-surface structures, particularly proteins and polysaccharides. Extracellular polysaccharides can be attached to cell membrane components as lipopolysaccharides (LPS) and lipoteichoic acids, they can form a capsule around the cell as capsular polysaccharides (CPS), or they can be released as exopolysaccharides (EPS) (Roberts, 1996). EPS produced by lactic acid bacteria (LAB) display a great variety of structures, and many are heteropolysaccharides composed of different sugar moieties (glucose, galactose, rhamnose, mannose, N-acetylglucosamine, N-acetylgalactosamine, glucuronic acid) (De Vuyst & Degeest, 1999; De Vuyst et al., 2001; Ricciardi & Clementi, 2000). In addition to their technological properties in fermented milk products (Ruas-Madiedo et al., 2002; van Kranenburg et al., 1999a), EPS produced by LAB may also have biological roles, such as immunomodulatory (Hosono et al., 1997), antitumour (Kitazawa et al., 1991) and cholesterol-lowering activities (Nakajima et al., 1992). The EPS from Lactobacillus rhamnosus RW-9595M stimulates interleukin, tumour necrosis factor and interferon gamma in mouse splenocytes (Chabot et al., 2001).

EPS production by Lb. rhamnosus varies greatly among strains (Dupont et al., 2000). The EPS production by strain RW-9595M (2350 mg l–1; Bergmaier et al., 2003) is among the highest measured to date for LAB, while strain ATCC 9595 produces from 50 to 116 mg l–1. Lb. rhamnosus RW-9595M produces an EPS with an exceptionally high rhamnose content, composed of heptasaccharide subunits of L-rhamnose, D-glucose and pyruvate-substituted D-galactose in a molar ratio of 4 : 2 : 1 (Van Calsteren et al., 2002). The structure of the repeating unit remains remarkably stable under different conditions for a number of Lb. rhamnosus strains (Van Calsteren et al., 2002). EPS with unusual monosaccharides such as L-rhamnose represent a source of new oligosaccharides and high-value substrates in the synthesis of pharmaceutical and aromatic compounds (Farres et al., 1997; Paul et al., 1986). As polysaccharide structure has a great influence on the technological properties and biological activities of EPS, rapid identification of new structures through genetic screening techniques will accelerate the discovery of LAB strains producing EPS with novel functional properties.

Biosynthesis of heteropolysaccharides starts with the intracellular formation of EPS precursors, the sugar nucleotides uridine-5'-diphosphate (UDP)-glucose, UDP-galactose and deoxythymidine diphosphate (dTDP)-rhamnose, which are the donor monomers for the biosynthesis of most repeating units. Genes directing heteropolysaccharide biosynthesis in LAB have been sequenced for a number of genera and species (Broadbent et al., 2003; Jolly et al., 2001, 2002b; van Kranenburg et al., 1997). Genetic elements required for EPS production include genes encoding regulation, chain-length determination, repeat-unit assembly, polymerization and export (De Vuyst & Degeest, 1999). In spite of accumulating knowledge of EPS gene organization, very little is known about the mechanism regulating EPS biosynthesis.

Despite their economic importance, few genetic studies of EPS production in Lactobacillus species have been reported (Jolly et al., 2002a; Lamothe et al., 2002). The objective of this work was to compare the genetic organization of the EPS biosynthesis gene cluster among strains of Lb. rhamnosus that vary with respect to their stable capacity for rhamnose-containing EPS production. Comparison of gene structure, sugar precursor biosynthesis and regulatory elements among strains is essential to understand the basis of the diversity in EPS production levels and composition. Our study reveals that the Lb. rhamnosus EPS gene organization differs considerably from other LAB, including other lactobacilli, but shows remarkably little variation among the four strains examined. In order to facilitate comparative studies on genes encoding bacterial surface polysaccharides, we have adopted the bacterial polysaccharide gene nomenclature system, which assigns the same symbol to homologous genes according to their function (Broadbent et al., 2003; Reeves et al., 1996).


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and media.
All bacterial strains and plasmids are listed in Table 1. Lb. rhamnosus cultures were incubated at the optimal growth temperature of 37 °C (Dupont et al., 2000) in MRS broth (Quelab) (De Man et al., 1960). Escherichia coli strains were grown in Luria–Bertani (LB) medium (Miller, 1992) at 37 °C with aeration. When necessary, media were solidified with 1·5 % agar (Quelab). All stock cultures were maintained at –80 °C in growth medium or BHI supplemented with 15 % (w/v) glycerol. Viable lactobacilli counts were determined by spread-plating 10-fold serial dilutions (in 0·1 %, w/v, sterilized peptone water) on MRS agar, and incubating aerobically at 37 °C for 48 h.


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Table 1. Source and relevant characteristics of strains and plasmids used in this study

 
Production, purification and quantification of EPS.
Lb. rhamnosus strains ATCC 9595 and RW-9595M were selected in order to represent low and high EPS production levels, respectively. Fermentations were carried out in triplicate in 2 l fermenters (Biostat M; B. Braun) with 1·6 l BMM medium (Morishita et al., 1981) containing 2 g tryptone l–1 and 40 g glucose l–1. The pH was adjusted to 6·0 with HCl, and maintained by the addition of 7 M NH4OH. After inoculating at 1 % (v/v) with a 16 h preculture in MRS medium, incubation was carried out at 37 °C under constant agitation of 100 r.p.m. Samples of 50 ml were taken at 0, 16, 20, 24 and 40 h. Growth was measured by serial dilution plating on MRS agar, and by OD600 measurement. An OD600 of 1·0 represents a dry mass (cells and attached EPS) of 1·4 g l–1 for ATCC 9595, and 2·1 g l–1 for RW-9595M.

EPS isolation and purification were carried out by ethanol precipitation, as described by Cerning et al. (1994), with a few modifications. Samples were heated for 15 min at 100 °C, then cells were eliminated by centrifugation at 12 000 g for 45 min at 4 °C. EPS were precipitated from the supernatant with 3 vols 95 % ethanol at 4 °C for 16 h, and collected by centrifugation at 12 000 g for 20 min. EPS pellets were dissolved in deionized water, and dialysed over a period of 3 days at 4 °C, with two water changes per day. Total sugars were measured by the phenol/sulfuric acid method (Dubois et al., 1951), with glucose as a standard, and the results are expressed in mg glucose l–1.

DNA isolation and manipulation.
Lb. rhamnosus cells from 16–18 h cultures (2 or 10 ml) were harvested by centrifugation (8000 g, 10 min), and DNA was extracted as described previously (Péant & LaPointe, 2004; Vincent et al., 1998). PCR was performed using standard conditions (Ausubel, 1995) with Taq polymerase (Promega) and the primers listed in Table 2. Platinum Pfx DNA polymerase (Invitrogen Life Technologies) was used to amplify larger fragments (>2 kb). PCR products were purified using the QIAquick PCR Purification Kit (Qiagen).


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Table 2. Primers

 
For chromosome walking, the Universal GenomeWalker kit was used according to the manufacturer's recommendations (BD Biosciences). For inverse PCR experiments, 200 ng DNA was digested with rare-cutting restriction enzymes, purified, and ligated using T4 DNA ligase (Boehringer Mannheim) for 10 h at 16 °C. The amplification reaction was carried out using the rTth DNA polymerase (Applied Biosystems) in a reaction volume of 100 µl. PCR cycling was done with the following conditions: 92 °C for 2 min, 30 cycles of 92 °C for 45 s, 60 °C for 1 min, and 68 °C for 4 min, followed by 6 min at 68 °C, and holding at 16 °C. Amplifications were visualized on a 0·8 % agarose gel after ethidium bromide staining by UV transillumination (Gel-Doc 1000; Bio-Rad). PCR amplicons less than 4 kb were cloned into pCR 4-TOPO, while those 4 kb and over were cloned into pCR XL-TOPO, according to the manufacturer's recommendations (Invitrogen).

Nucleotide sequencing and sequence analysis.
The DNA sequence of both strands was determined by the sequencing service of Université Laval (Life and Health Sciences Pavilion) using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems), with either universal primers or the primers listed in Table 2. DNA sequence analysis and similarity searches were carried out using the BLAST network service at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/), National Institutes of Health (Altschul et al., 1997). BLASTX software (http://www.ncbi.nlm.nih.gov/blast/) was used to conduct similarity searches of the nucleotide and protein databases. Membrane-spanning regions of translated gene products were predicted using the TMpred program (http://www.ch.embnet.org/software/TMPRED_form.html).

Gene nomenclature.
Genes were named according to the bacterial polysaccharide gene nomenclature (BPGN) system (www.microbio.usyd.edu.au/BPGD/default.htm). This nomenclature provides a single name for genes of a given function, and it can be applied to all species (Reeves et al., 1996). The BPGN system has not been used widely for LAB, except for some Streptococcus thermophilus strains (Broadbent et al., 2003). The designation ‘we*’ is used for naming genes involved in EPS biosythesis, while ‘wc*’ is used for capsule genes. The designation ‘wz*’ was proposed for genes with homologous predicted functions in regulating polysaccharide processing and polymerization; these genes are found in many polysaccharide gene clusters. Finally, genes implicated in nucleotide sugar precursor biosynthesis are named for the pathway, such as ‘rml’ for the dTDP-L-rhamnose pathway.

Analysis of gene transcription.
Total cellular RNA was extracted from 10 ml cultures of Lb. rhamnosus strains ATCC 9595 and RW-9595M after 6 h (exponential phase) or 24 h (stationary growth phase) incubation by using the RNeasy Midi Kit (Qiagen). All RNA isolation steps were performed according to the manufacturer's instructions, except that 50 U mutanolysin ml–1 (Sigma) was used in addition to lysozyme to degrade the cell walls, and incubation time was extended to 1 h. The isolated RNA was treated with RNase-free DNase I (Qiagen) at 25 °C for 1 h, followed by a second purification using an RNeasy column.

For RT-PCR (One-step kit; Qiagen), reverse transcription was performed with primers (Table 2) derived from the Lb. rhamnosus ATCC 9595 EPS biosynthesis locus to obtain an amplicon length between 1·5 and 3 kb. As positive controls, each region was amplified with the same primers using chromosomal DNA of Lb. rhamnosus ATCC 9595 as the template. As negative controls, reaction product without the reverse transcription stage was used as the template. Identification of the 5' end of the EPS gene-specific mRNA, and characterization of putative promoter activities, were carried out using the 5'-RACE kit (Invitrogen Life Technologies) and the primers listed in Table 2. All experimental steps were performed according to the manufacturer's instructions. This method captures the 5'-end information of mRNA through synthesis of first-strand cDNA initiated from a gene-specific reverse primer. An anchor sequence was then added to the 3' end of the cDNA using terminal deoxynucleotide transferase, followed by direct amplification of tailed cDNA using the nested gene-specific primers and the anchor-specific primer provided. PCR products were cloned and sequenced as described above.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Growth and EPS production by strains ATCC 9595 and RW-9595M
Mean specific growth rates (h–1) were 0·266±0·006 for ATCC 9595, and 0·296±0·027 for RW-9595M. These values are comparable between strains, considering the precision limits of the plate-count method. Glucose was exhausted in the medium after the total incubation period (Fig. 1). RW-6541M produced less EPS than ATCC 9595, whereas strain R exhibited an EPS production between those of RW-9595M and ATCC 9595 (Table 1). The maximum EPS production at 40 h incubation was 1611 mg l–1 for strain RW-9595M, while most of the EPS production occurred by 24 h (Fig. 1). For ATCC 9595, the maximum production of 116 mg l–1 was attained after 40 h incubation. Strain R produced a maximum of 498 mg l–1 after 24 h incubation, while strain RW-6541M produced only 61 mg l–1 after 72 h incubation.



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Fig. 1. EPS production ({blacklozenge}), residual glucose ({blacksquare}) and lactic acid accumulation ({blacktriangleup}) during incubation of Lb. rhamnosus RW-9595M (a) and ATCC 9595 (b) in BMM medium containing 40 g glucose l–1 controlled at pH 6·0.

 
Cloning and sequencing of the EPS biosynthesis loci from four Lb. rhamnosus strains
Specific nested primers (Table 2) were designed from the C-terminal region of the priming glycosyltransferase gene catalysing the first step in polysaccharide synthesis for strain ATCC 9595 (Provencher et al., 2003) (GenBank accession no. AF323527). These primers were used for isolating one PCR product covering 3·5 kb upstream of the priming glycosyltransferase (welE) and 1·5 kb downstream. Inverse PCR was subsequently used to obtain the complete coding region of the EPS gene locus of Lb. rhamnosus ATCC 9595. Alignment of the DNA sequences obtained from the amplicons resulted in an 18·7 kb contiguous sequence (Fig. 2). Equally spaced primers (Table 2) were then designed along the length of this sequence for amplification and cloning of five fragments of about 4 kb each from strains RW-9595M, R and RW-6541M.



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Fig. 2. Organization of the EPS biosynthesis gene cluster from Lb. rhamnosus strains RW-9595M, ATCC 9595, R and RW-6541M. Promoters (numbered from P1 to P5) and putative terminators are represented as flags and hairpins, respectively. Arrows represent potential ORFs and gene designations are indicated under the arrows. Grey arrows indicate ORFs involved in dTDP-rhamnose precursor biosynthesis.

 
The DNA sequence of 18·7 kb was completely determined on both strands. The G+C content of this region varied from 41·6 (strain R) to 42·1 mol% (RW-9595M), which is close to the typical 42–48 mol% G+C content reported for the Lb. rhamnosus genome (Sriranganathan et al., 1985).

Sequence analysis and comparison
ORF analysis revealed the presence of 18 putative ORFs (Fig. 2). Overall pairwise comparison revealed that the four DNA regions are highly homologous, at 99 %. RW-9595M and ATCC 9595 have 18 bp different out of 18 749 bp, while RW-6541M and R show a difference of 34 out of 18 750 bp. Between strains of these two groups, there are 139–150 bp different. Upstream of the first ORF for 456 bp, the DNA sequences are 100 % identical for ATCC 9595 and RW-9595M, as well as for RW-6541M and R. Between these two groups, this segment is 98 % identical.

Of the predicted gene products, 17 are similar to proteins involved in the biosynthesis of various bacterial polysaccharides (Table 3). Based on these sequence similarities, a putative biological function could be attributed to most of the predicted proteins. Within the gene region encoding EPS biosynthesis, one ORF (orf1) encodes a truncated transposase of the IS1165 family (Johansen & Kibenich, 1992). All ORFs were in the same orientation except for orf1 and wzr (Fig. 2).


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Table 3. Profiles of predicted ORFs of the EPS biosynthesis gene locus of Lb. rhamnosus RW-9595M

 
In the 5' region, the first two ORFs (wzd and wze) have predicted amino acid sequences that most resemble proteins involved in putative polysaccharide-chain-length determination in Leuconostoc mesenteroides (39 % for Wzd versus Wzz) and Lactobacillus delbrueckii subsp. bulgaricus (Lb. bulgaricus; 42 % for Wze versus EpsC; Lamothe et al., 2002). Prediction of membrane-spanning regions shows two putative transmembrane helices for Wzd [amino acids 18 (inside)–36 (outside) and amino acids 219 (outside)–238 (inside)]. The comparative analysis of Wze with various prokaryotic proteins known to autophosphorylate on tyrosine shows a Walker A [(AG)X4GK(ST)] ATP-binding motif (Walker et al., 1982), two Walker B (hhhD, where h represents a hydrophobic amino acid) magnesium-binding motifs and a series of tyrosine residues, termed the tyrosine cluster, located in the C-terminus. In the 3' region of the cluster, gene wzb is located 154 bp downstream of wzr. Wzb has homology with the phosphotyrosine-protein phosphatase EpsD from Lb. bulgaricus (Lamothe et al., 2002) and CpsB from Streptococcus pneumoniae, and is also proposed to be involved in chain-length determination (Morona et al., 2002). The three gene products (wzd, wze and wzb) show 98–100 % identity among the four Lb. rhamnosus strains examined (Table 4), while the 19 bp intergenic region between wzd and wze is also identical.


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Table 4. Comparison of the predicted amino acid sequences of ORFs of the EPS gene clusters of four strains of Lb. rhamnosus

The number of different amino acids is given in parentheses.

 
The central portion of the locus is occupied by five of the six genes (welF, welG, welH, welI, welJ and welE) encoding potential glycosyltransferases (Tables 4 and 5). The welE gene is located farther along in the 3' region, apart from the other glycosyltransferase genes, and displays 56 % identity with the predicted product Lp_1233 from Lactobacillus plantarum (Kleerebezem et al., 2003). A variable degree of identity (38–100 %) was found between the WelE predicted product and the undecaprenyl-phosphate glycosyl-1-phosphate transferase (also known as the priming glycosyltransferase) found in several capsular and EPS-producing bacteria. The function of the priming glycosyltransferase has been demonstrated for EpsD from Lactococcus lactis (van Kranenburg et al., 1999b). The predicted product of welF shows closest identity (30 % amino acids) to a putative glycosyltransferase from Bacteroides thetaiotaomicron (Xu et al., 2003), while WelH and WelI show moderate identity with rhamnosyltransferases involved in LPS biosynthesis in Shigella flexneri SflI (28 % for WelH versus RfbF) (Wei et al., 2003) and Lactococcus lactis subsp. lactis strain IL1403 (31 % for WelI versus RgpB) (Bolotin et al., 2001), as well as to those involved in capsule biosynthesis by St. pneumoniae (Morona et al., 1997), and in EPS biosynthesis by St. thermophilus CNRZ368 (Bourgoin et al., 1999). According to the sequence alignments, WelF, WelH and WelI are members of the glycosyltransferase family 2 (Table 5) (Campbell et al., 1997). WelG has the highest identity (24 % identical amino acids) with a putative glycosyltransferase from E. coli K-12 (Blattner et al., 1997). WelJ exhibits most identity (33 % amino acids) with glycosyltransferase Cps1B from Lb. plantarum WCSF1 (Kleerebezem et al., 2003), but also shows moderate homology with galactosyltransferase gene products found in S. thermophilus strains with different eps operon types (31 % with Eps3M, type III eps operon; GenBank accession no. AAL23736. WelG and WelJ are predicted to be members of the glycosyltransferase family 1 (Table 5) (Campbell et al., 1997). All six putative glycosyltransferases could therefore be involved in the sequential biosynthesis of the repeating unit. The glycosidic linkage catalysed by each enzyme can be proposed according to the structure of the previously determined heptasaccharide (Fig. 3, Table 5). Among the four strains examined, there are few amino acid differences in the glycosyltransferase sequences (Table 4). Some of these changes result in a conserved amino acid (such as Ile to Leu or Val), and will not be detailed here. There are three instances of loss of an Asp or a Glu, residues that have been proposed as catalytic residues in glycosyltransferase sequences (Campbell et al., 1997; Kapitonov & Yu, 1999). Strain R shows one amino acid difference in two glycosyltransferase gene products (Asp to Asn in WelG, and Glu to Gly in WelJ), while RW-6541M shows one amino acid change (Asp to Gly in WelI) in comparison with the corresponding sequences from the other three strains.


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Table 5. Properties of the proposed products encoded by the glycosyltransferase genes

 


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Fig. 3. Structure of the repeating unit of the EPS produced by Lb. rhamnosus RW-9595M and R (Van Calsteren et al., 2002). Gene products predicted to carry out the incorporation of each monosaccharide residue are indicated. Glc, glucose; Rha, rhamnose; Gal, galactose; p, pyranose.

 
Gene wzm could code for a pyruvyltransferase, as the predicted protein product has 16 % overall amino acid identity with a putative combined pyruvyltransferase/O-methyltransferase (DVUA0070) from Desulfovibrio vulgaris subsp. vulgaris (Heidelberg et al., 2004). Wzm shows 27 % identity over the N-terminal 300 aa of DVUA0070. Among the four strains, Wzm shows 98–100 % identity (Table 4).

Gene wzx is located 377 bp downstream of wze in the 5' region of the locus, and this 379 bp intergenic region is 98·6 (five nucleotides different) to 100 % identical among the four strains. The predicted amino acid sequence of wzx displays moderate identity (31 % amino acids) with Wzz from S. thermophilus strain MR-2C (Broadbent et al., 2003). Similar proteins found in eps gene clusters from LAB are predicted to act as the EPS repeat-unit transporter, such as EpsM from S. thermophilus Sfi6, for example (Stingele et al., 1996). Prediction of membrane-spanning regions shows 13 putative transmembrane helices (data not shown). Wzx is 98 % identical between RW-9595M, ATCC 9595 and RW-6541M, whereas the same gene product from strain R shows only 92 % identity with the other strains, because of a point mutation (G to A) inserting a stop codon which truncates the final 34 aa. Wzy has nine highly probable transmembrane helices, and shows some similarity (20 % identity) with the putative polysaccharide polymerase Eps11O from the S. thermophilus type XI eps operon. Wzy is 100 % identical among the four strains. The 16 bp intergenic region between wzy and welJ is also 100 % identical.

Four genes (rmlArmlD) were found in the central region of the locus, 191 bp downstream of wzm, and 826 bp upstream of welE. Considering the high degree of identity (Table 3), the predicted functions of these four gene products are respectively glucose-1-phosphate thymidylyltransferase (also known as dTDP-glucose pyrophosphorylase), dTDP-D-glucose 4,6-dehydratase, dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase and dTDP-6-deoxy-L-lyxo-4-hexulose reductase (Table 3). These enzymes would be responsible for the production of the rhamnose precursor essential for EPS biosynthesis. Southern hybridization analysis using these four genes as a probe revealed that the Lb. rhamnosus genome has only one copy of these genes (data not shown). Among the four strains, the four gene products are 98–100 % identical (Table 4). The intergenic regions of rmlAC (14 bp) as well as rmlBD (61 bp) are 100 % identical among the four strains.

In the 3' region of the EPS locus, gene wzr is organized in the opposite transcriptional sense. The predicted product Wzr shows closest identity (46 %) to the putative transcription regulator Ooen02000936 from Oenococcus oeni, and shows 31 % identity with EpsA from Lb. bulgaricus (cell-envelope-related transcriptional attenuator; GenBank accession no. AAG44705 (Lamothe et al., 2002). The wzr sequence from RW-9595M differs from the remaining three strains by a Pro residue in position 44 instead of Ser, and Phe instead of Leu in position 139. As these changes in amino acids could result in changes in protein secondary structure (for phenylalanine) or post-translational modification (Ser phosphorylation), they may have an impact on the function of this potential regulator. A short interrupted ORF, designated orf1, is located just upstream of wzr. The translated product of orf1 is similar to a transposase of Leuconostoc mesenteroides subsp. cremoris IS1165 (38 % identity over 94 aa of translated orf1) (Johansen & Kibenich, 1992).

Transcriptional analysis of the EPS biosynthesis gene cluster
Analysis of the DNA sequence suggests that the EPS gene cluster of Lb. rhamnosus is organized into five transcriptional units (Fig. 2). Five putative promoters with –35 and –10 sequences were identified with 23, 50, 63, 33 and 25 bp spacing between the ends of the –10 sequence and the start codon of ORFs wzd, rmlA, welE, wzr and wzb, respectively. For each of the five promoters, the sequences were 100 % identical among the four Lb. rhamnosus strains. Overlapping reading frames could be identified at the end of wzx and the beginning of welF (4 bp overlap), at the end of welG and the beginning of welH (11 bp overlap), at the end of welH and the beginning of welI (20 bp overlap), and finally at the end of welI and the beginning of wzy (1 bp overlap). This analysis suggests the transcriptional coupling of these six genes. Putative rho-independent transcription terminator sequences were identified downstream of rmlD (–17·6 kcal mol–1, –73·6 kJ mol–1), welE (–12·2 kcal mol–1, –51·0 kJ mol–1) and wzb (–29·5 kcal mol–1, –123·4 kJ mol–1). The putative terminator of welE could also be the putative terminator of wzr, which is transcribed in the opposite sense. To confirm some of these hypotheses, total RNA was isolated from ATCC 9595 at two different growth phases (OD600 0·6 and 3), and used for RT-PCR and 5' RACE experiments.

Successive overlapping RT-PCR reactions were carried out to determine the whether the EPS genes are co-transcribed in an identical fashion for strains ATCC 9595 and RW-9595M. Specific amplifications were obtained for each RT-PCR carried out (Fig. 4), indicating that all genes from wzd to welE (inclusive) are transcribed as a single 15·4 kb polycistronic mRNA from a promoter sequence upstream of wzd. In particular, an amplification fragment was obtained by the RT-PCR which covered the 191 bp intergenic region between wzm and rmlA (Fig. 4c, lane 5). This region is identical between RW-9595M and ATCC 9595, and varies by only 3 or 4 nucleotides from the same region in strains RW-6541M and R. Another amplified fragment covered the region from rmlD to welE (Fig. 4a and b, lane 4). The 827 bp intergenic space varied by 3 nucleotides (between RW-9595M and ATCC 9595) to 5 nucleotides (RW-9595M with R and RW-6541M). In the 3' region, two single-gene (wzr and wzb) RT-PCR reactions gave positive results, but no amplification was obtained in this region when two or mores genes were combined (data not shown). This 157 bp intergenic region varied by 0–2 nucleotides among the four strains.



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Fig. 4. Transcription analysis by RT-PCR of EPS genes of Lb. rhamnosus strains ATCC 9595 (a) and RW-9595M (b) carried out on RNA isolated after incubation for 6 h at 30 °C. Lanes: 1, the 1 kb ladder (Invitrogen); 2, wzd–welF; 3, rmlArmlD; 4, rmlD–welE; 5, welE alone; 6, negative control (PCR without the RT step). (c) RT-PCR of wzdwelE transcripts from Lb. rhamnosus ATCC 9595. Lanes: 1, wzd–wzx; 2, is wzxwelG; 3, welGwelI; 4, welIwelJ; 5, welJrmlA; 6, rmlArmlB; 7, rmlBrmlD; 8, negative control; 9, welE. Lane M: 1 kb ladder (Invitrogen Life Technologies).

 
Determination of transcriptional start sites
Experiments by 5' RACE were performed on total RNA isolated from the early stationary growth phase of a Lb. rhamnosus ATCC 9595 culture. The transcription start of each of the five putative promoters was detected in front of the putative RBS site using antisense oligonucleotides derived from the 5' sequence of wzd, rmlA, welE, wzr and wzb (Table 2). Upstream of each transcription start, putative consensus sequences for the –35 and –10 regions were located (Fig. 5), and these are in good agreement with the consensus sequence of vegetative promoters identified in Lb. rhamnosus (McCracken et al., 2000). Conserved bases corresponding to the consensus AT-rich UP element (upstream enhancer element; Estrem et al., 1998) were identified upstream of the –35 hexamer of each putative promoter.



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Fig. 5. Comparative analysis of nucleotide sequences and transcriptional start sites of promoters located in the EPS biosynthesis locus from Lb. rhamnosus ATCC 9595. Sequences of –35 and –10 hexamers are underlined and compared with their respective consensus sequences GxTTraca and TAtaaT (T>75 %, T 60–75 %, t 40–59 %, r represents adenosine or guanosine, and x represents any nucleotide) (McCracken et al., 2000). Transcription start sites are indicated under +1 as underlined single bold A or G nucleotides. Dots are used for alignment of –10 hexamer sequences and +1 start sites. The sequence similar to CRP (Gaston et al., 1989) is boxed.

 
The putative P1 promoter region is at the appropriate distance from the transcriptional starting point determined by 5' RACE (Fig. 5). Analysis of the region upstream of the first promoter, P1, of the Lb. rhamnosus EPS gene clusters reveals three significant motifs. First, a sequence was found almost 200 bases upstream of promoter P1 that differed by two nucleotides (ATAAAAGTTTTACA) from the cre site (ATAAAACGTTTACA) of the lactose operon of Lactobacillus casei (Gosalbes et al., 1997). Secondly, an UP element (Estrem et al., 1998) is positioned at one DNA turn further upstream than in the other Lactobacillus spp. promoters identified to date (McCracken et al., 2000). Third, a putative regulation site was identified overlapping the –35 hexamer. A symmetrical TGA half-site motif is localized 5 bp upstream of the –35 hexamer, and a TCA half-site motif is localized in the –35 hexamer. This motif (TTTGActagctTCACA) displays only one nucleotide difference from the CRP consensus sequence of E. coli (TGTGANNNNNNTCACA) (Gaston et al., 1989), some deviation from the FLP consensus sequence of Lb. casei (CA/CTGANNNNTCAG/TG) (Gostick et al., 1998), and three nucleotides different from the FNR consensus sequence from E. coli (TTGATNNNNATCAA) (Spiro & Guest, 1990).


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
This is the first comparative analysis of EPS-related genes for Lb. rhamnosus, to our knowledge. Lb. rhamnosus strain RW-9595M has been shown to produce up to 2350 EPS mg l–1 after 32 h of pH-controlled (pH 6) growth at 37 °C in supplemented whey permeate medium (Bergmaier et al., 2003), which is the highest quantity recorded to date for heteropolysaccharide production by LAB. This strain is a spontaneous variant of Lb. rhamnosus ATCC 9595. There is very low probability that the EPS produced by Lb. rhamnosus ATCC 9595 differs in structure from the EPS secreted by the variant (M. R. Van Calsteren, personal communication), especially given the high conservation in glycosyltransferase gene products among the strains examined. The presence of a few amino acid differences does not have an effect on enzyme specificity, as the same EPS structure is produced by strains RW-9595M, R (Van Calsteren et al., 2002) and RW-6541M (M. R. Van Calsteren, personal communication). There is thus a high correlation between glycosyltransferase gene presence and EPS repeat-unit structure that can be used in future screening of Lb. rhamnosus strains for novel EPS structures. The variation in EPS production levels observed among the Lb. rhamnosus strains studied here may originate from differences in central metabolism and the availability of sugar precursors. Studies are presently under way to correlate differences in EPS production among Lb. rhamnosus strains with differences in the activity of enzymes of central metabolism and precursor biosynthesis.

The genetic organization and transcription of the EPS gene clusters from Lb. rhamnosus show very important differences from those of the gene clusters involved in the synthesis of capsular and free EPS in Gram-positive bacteria such as S. thermophilus (Stingele et al., 1996), Lactococcus lactis subsp. cremoris (van Kranenburg et al., 1997), Lb. bulgaricus (Lamothe et al., 2002) and Lactobacillus helveticus (accession no. AX009415) (Germond et al., 1999). Fifteen genes are co-transcribed on one long mRNA, as has been reported for other EPS biosynthesis gene clusters. However, transcription of the EPS genes of Lb. rhamnosus may be driven by five different promoters, as five start sites were determined. Although these start sites are appropriately spaced from promoter sequences, there is a possibility that they may be formed by mRNA processing. This indicates the potential for a complex system for regulating the expression of these genes in Lb. rhamnosus.

Six genes which probably encode the glycosyltransferases responsible for the structural organization of the heptasaccharide repeating unit are co-transcribed with genes involved in the polymerization and export of the EPS. In all EPS biosynthesis gene clusters reported to date, the priming glycosyltransferase gene is located just upstream of all other glycosyltransferase genes. In the Lb. rhamnosus strains examined, the welE gene encoding the first glycosyltransferase is located after the other glycosyltransferase genes, but is also transcribed from its own promoter, P3. This suggests an independent expression of the priming glycosyltransferase that may lead to higher amounts of mRNA, and thus to an improved initiation of EPS repeat-unit biosynthesis through the translation of more priming glycosyltransferase proteins.

The carbohydrate linkage arrangement of the heptasaccharide repeating unit from Lb. rhamnosus strains R and RW-9595M was determined previously (Van Calsteren et al., 2002). The order of the monosaccharides represents the proposed sequential addition of sugars during biosynthetic assembly of the repeating unit (Fig. 3), beginning with WelE, which transfers the first glucose onto the lipophilic carrier molecule. The glycosyltransferases carrying out the next reactions form an ordered biosynthetic complex that reflects the order of the genes. Only three putative rhamnosyltransferases (WelF, WelH and WelI) were found for the addition of four rhamnose residues to the repeating unit. We hypothesize that the same rhamnosyltransferase (WelI) could accomplish the addition of two rhamnose residues in the fifth and sixth positions by catalysing the formation of identical {alpha}(1->3) glycosidic linkages. In fact, previous studies reported two mannosyltransferases (MtfA and MtfB) from E. coli that transfer mannose residues, respectively forming two {alpha}(1->3) and three {alpha}(1->2) bonds in the O9 antigen biosynthesis pathway (Kido et al., 1995). Once the hexasaccharide of the backbone is assembled, WelJ, the {alpha}(1->2) galactosyltransferase, could add one branching sugar to form the final heptasaccharide repeat unit. Finally, Wzm may be the pyruvyltransferase that adds the pyruvate substituent to this galactose residue.

Genes involved in chain-length determination are not organized at the beginning of the cluster, but are divided into two regions: wzd and wze are in the 5' region of the cluster, while wzb is located at the end of the cluster. Given the similarity in predicted protein products of these three genes with other gene products involved in chain-length determination, the model of regulating EPS biosynthesis at the post-transcriptional level described for S. pneumoniae (Bender et al., 2003; Morona et al., 2000, 2003) is probably applicable to a certain degree to Lb. rhamnosus as well. All CpsD-specific motifs and residues involved in the regulating model of S. pneumoniae were also found in Wze (Walker A and Walker B ATP-binding motifs, tyrosine cluster). These motifs are usually found in protein kinases involved in the biosynthesis of EPS by Gram-negative and Gram-positive bacteria. Bender et al. (2003) showed that deletion of cps2C or cps2D led to short-chain polymers instead of long polysaccharide molecules in S. pneumoniae.

Four genes (rmlArmlD) exhibit significant similarity to rml gene products involved in the anabolism of dTDP-L-rhamnose from {alpha}-D-glucose 1-phosphate. EPS biosynthesis gene clusters of LAB do not generally contain these four genes, while cps clusters often do (Morona et al., 1997). For S. pneumoniae, rml gene expression seems to be controlled by the promoter of the cps cluster (Iannelli et al., 1999). For Lb. rhamnosus, we observed that the transcription of these four genes could be controlled by two different promoters, as they are cotranscribed on a large 15·2–15·4 kb mRNA from wzd to welE, and also independently from promoter P2. Expression of rmlAD genes might be activated independently of the EPS biosynthesis genes to form the cell wall polysaccharides, but cotranscription of these genes with the EPS biosythesis gene cluster might increase the dTDP-L-rhamnose pool available for biosynthesis of EPS. Location of genes involved in rhamnose precursor biosynthesis within this locus, and their coordinated expression, suggest the importance of this monosaccharide for biosynthesis of the Lb. rhamnosus EPS structure, where four out of seven monosaccharides of the repeat unit are rhamnose.

The four Lb. rhamnosus strains produce very different quantities of EPS under the same conditions (from 61 to 1611 mg l–1), yet the gene clusters show remarkably little variation, even in all the promoter regions, including the region upstream of promoter P1. The potential cre sequence found upstream of P1 shows significant differences from the consensus cre site (TGWNANCGNTNWCA) (Weickert & Chambliss, 1990), the target of the CcpA regulator mediating catabolite repression and carbon flow in Gram-positive bacteria (Henkin, 1996). The central CG nucleotides of the cre site are essential for interacting with the CcpA complex (Weickert & Chambliss, 1990), but are degenerated in the sequence found upstream of the P1 promoter. As a consequence, catabolite repression does not appear to be a likely mechanism for regulating gene expression of the Lb. rhamnosus EPS biosynthesis locus.

In conclusion, comparative analysis of EPS biosynthesis gene clusters from strains that produce different EPS levels is the first step to providing new insight into this industrially relevant phenotype. Lb. rhamnosus strains show remarkable variation in EPS production levels, but little variation in EPS gene sequences. The presence of six genes encoding potential glycosyltransferases responsible for the structural organization correlated well with the assembly of the heptasaccharide repeating unit structure previously determined by our team. In addition, four genes were identified within the EPS biosynthesis locus that might result in coordinated production of the rhamnose precursor with the enzymes involved in EPS biosynthesis. Future studies will aim for functional analysis of the EPS gene products and their interactions.


   ACKNOWLEDGEMENTS
 
The authors thank the Natural Sciences and Engineering Research Council of Canada (Research Partnerships Program–Research Network on Lactic Acid Bacteria), Agriculture and Agri-Food Canada, Novalait of Quebec, Dairy Farmers of Canada and the Institute Rosell for financial support. We gratefully acknowledge Dr M. Frenette and Dr E. Emond for valuable contributions and discussions, as well as I. Pelletier for technical assistance. The authors are also grateful to A. Parent for carrying out EPS production and quantification, and to C. Provencher for contributing to this project from the beginning.


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Received 22 December 2004; revised 11 February 2005; accepted 24 February 2005.



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