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
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ABSTRACT |
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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).
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INTRODUCTION |
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EPS production by Lb. rhamnosus varies greatly among strains (Dupont et al., 2000). The EPS production by strain RW-9595M (2350 mg l1; Bergmaier et al., 2003
) is among the highest measured to date for LAB, while strain ATCC 9595 produces from 50 to 116 mg l1. 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
).
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METHODS |
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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 l1.
DNA isolation and manipulation.
Lb. rhamnosus cells from 1618 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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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 ml1 (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.
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RESULTS |
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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 139150 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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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 98100 % 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 mol1, 73·6 kJ mol1), welE (12·2 kcal mol1, 51·0 kJ mol1) and wzb (29·5 kcal mol1, 123·4 kJ mol1). 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 02 nucleotides among the four strains.
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DISCUSSION |
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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
(1
3) glycosidic linkages. In fact, previous studies reported two mannosyltransferases (MtfA and MtfB) from E. coli that transfer mannose residues, respectively forming two
(1
3) and three
(1
2) bonds in the O9 antigen biosynthesis pathway (Kido et al., 1995
). Once the hexasaccharide of the backbone is assembled, WelJ, the
(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 -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 15·215·4 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 l1), 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.
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ACKNOWLEDGEMENTS |
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Received 22 December 2004;
revised 11 February 2005;
accepted 24 February 2005.
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