Unité d'Ecologie et de Physiologie du Système Digestif, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy-en-Josas, France
Correspondence
Jamila Anba-Mondoloni
jamila.anba{at}jouy.inra.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY307023.
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INTRODUCTION |
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-Glucuronidase activity increases the enterohepatic circulation of toxic compounds. It thus plays a major role in the generation of toxic and carcinogenic metabolites which may promote tumour formation at different sites, including the large bowel (McBain & Macfarlane, 1998
; Arimochi et al., 1999
). Results from two different studies (Hawkesworth et al., 1971
; Kim et al., 1998
) suggest that
-glucuronidase activity can be considered a cancer-risk biomarker.
On the other hand, -glucuronidases are also capable of selectively activating low-toxicity glucuronide prodrugs into highly cytotoxic agents at the tumour site, leading to a better anti-tumour effect and a reduction of systemic toxicity (De Graaf et al., 2002
; Chen et al., 2003
). In addition, these enzymes may play a beneficial role by releasing aglycone residues with protective effects, such as lignans, flavonoids, ceramide and glycyrrhetinic acid. These molecules are active against tumours, platelet aggregation, viral infection, and allergic or inflammatory responses. Some of them have antioxidant effects (Kim et al., 1998
; Jenab et al., 1999
; Schmelz et al., 1999
; Akao, 2000
; Kim et al., 2000
; Jeong et al., 2002
; Kohno et al., 2002
).
-Glucuronidase activity has been detected among bacterial genera belonging to the dominant human intestinal microbiota, such as Bacteroides, Bifidobacterium, Eubacterium and Ruminococcus (Hawkesworth & Hill, 1971
; McBain & Macfarlane, 1998
; Akao, 1999
, 2000
). Among bacteria, genes encoding
-glucuronidase have been described for Escherichia coli, Lactobacillus gasseri and Staphylococcus sp. (Jefferson et al., 1986
; Jefferson, 1989
; Russel & Klaenhammer, 2001
), and identified in Clostridium perfringens, Staphylococcus aureus and Thermotoga maritina, for which whole genome sequences are available (Jefferson et al., 1986
; Nelson et al., 1999
; Russel & Klaenhammer, 2001
; Shimizu et al., 2002
). Previous studies have shown that
-glucuronidase is found in some bacteria of the gastrointestinal tract, such as Eubacterium and Bacteroides (Hill et al., 1971
; McBain & Macfarlane, 1998
; Akao, 1999
, 2000
). However, no
-glucuronidase gene has been described in these bacteria and in other strict anaerobes from the digestive ecosystem.
Here, we describe the identification of a new -glucuronidase gene, gus, from Ruminococcus gnavus strain E1. R. gnavus E1 is a Gram-positive, strictly anaerobic strain isolated from the dominant human intestinal microbiota of a healthy donor (Ramare et al., 1993
; Dabard et al., 2001
). This strain is capable of expressing very high
-glucuronidase activity in culture. The nucleotide and deduced amino acid sequences of this gene are described. Additionally, its genetic environment is presented and its expression in R. gnavus E1 was investigated.
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METHODS |
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DNA sequencing and analysis.
Nucleotide sequences were determined on both strands using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer) and an ABI Prism 377 DNA automated sequencer (Perkin-Elmer). Nucleotide sequences were assembled with the Staden program (Hawkesworth et al., 1971) and analysed with GCG software (Wisconsin package version 10.3, Accelrys Inc., San Diego, CA). Criteria applied for identifying putative genes were the length of ORFs (>30 codons) and translation initiation signals (start codons ATG, TTG, GTG, ATA or ATC) preceded by a potential ribosome-binding site (RBS).
The GenBank accession number for this sequence is AY307023.
Construction of plasmids.
PCR fragments of the gus gene region encoding -glucuronidase in R. gnavus strain E1 were obtained using several pairs of primers (Table 2
) and were ligated into the pGem-T vector (Promega). Ligation mixtures were introduced by electroporation into the E. coli
uidA : : kanR strain (L91), described below, and selected on LB agar plates containing 100 µg ampicillin ml1.
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Enzyme assays.
-Glucuronidase activity in cell extracts was measured using a spectrophotometer to quantify the rate of release of p-nitrophenol (
=400 nm) from p-nitrophenyl-
-D-glucuronide (PNPG) (Sigma-Aldrich). Cultures (equivalent to 109 cells) were washed in 1 ml phosphate buffer (0·1 M sodium phosphate, pH 6·8). The cell pellet was resuspended in 1·6 ml of the same buffer. After adding 500 mg of 106 µm diameter glass beads (Sigma-Aldrich), cell suspensions were subjected to two cycles of 40 s at power 4·5 m s1 on a FastPrep FP120 instrument (BIO 101-Q.BIOgene), with 1 min on ice between cycles. The pellets, beads and cell debris were removed by centrifugation. The cell extracts were collected and kept temporarily on ice until the start of the assays. Protein concentrations were determined using the FolinLowry method (Lowry et al., 1951
). For each assay, the cell extracts were diluted to a quarter in 3 ml of phosphate buffer (0·1 M sodium phosphate, pH 6·8). These diluted cell extracts were warmed to 37 °C and 1·7 ml of 0·5 mM PNPG was added. At appropriate time intervals, usually 5, 10, 15, 30 and 45 min, the reaction was stopped by adding 1·6 ml of 125 mM Na2CO3 to 300 µl of the reaction mixture. Optical density was measured at 400 nm. One unit of activity was defined as 1 nanomole of p-nitrophenol liberated per minute per milligram of protein. Each value presented is the mean of the results from at least three independent measurements.
Southern hybridization.
To identify the longest genomic fragment carrying the gus gene and to select the enzyme to use for inverse PCR, R. gnavus E1 DNA was digested with EcoRV, PstI, NcoI, BglII and SmaI in independent reactions. Digested genomic DNA (10 µg) was separated by electrophoresis through a 1 % agarose gel (Tebu, Le-Perray-en-Yvelines, France) and transferred by capillary blotting to a Hybond-N+ nylon membrane (Amersham Biosciences). Membranes were hybridized with ECL-labelled oligonucleotides (Amersham Biosciences) complementary to the coding strand of the gus gene of strain E1. Signal detection was carried out as recommended by the supplier.
Inverse PCR.
EcoRV-digested DNA (10 µg) of R. gnavus E1 was circularized with a ligase kit, as recommended by the supplier (for enzyme selection, see Methods, Southern hybridization). PCR was performed with the 3R/4R primers, and the product obtained was cloned in pGem-T and sequenced using universal primers appropriate to the vector (#1211F and #1233Rev, Biolabs).
RNA extraction.
RNA extractions were performed following the instructions in the High Pure RNA Kit (Roche Diagnostics). Bacteria (2x109 cells) were briefly disrupted, in the presence of phenol, 10 % SDS, 3 M sodium acetate (pH 5·2) and glass beads (Sigma-Aldrich) by shaking using the Fast Prep centrifuge FP120 (BIO 101-Q.BIOgene), as described above. Cell lysates were obtained after a 15 min centrifugation and treated to isolate total RNA. The concentration and quality of the RNA were determined by measuring the A260 and A280 with a BIOphotometer (Eppendorf) and by agarose gel electrophoresis. Trace amounts of DNA in RNA samples were removed by treatment with DNase I (Invitrogen SARL).
RT-PCR analysis.
cDNA synthesis was performed by reverse transcription of 2 µg RNA primed with 500 ng random primer hexamer (New England Biolabs). The reaction was carried out at 37 °C for 50 min with M-MLV Reverse Transcriptase (Ambion), as recommended by the supplier. A dilution (1 : 20) of the cDNA solution obtained was used for PCR amplification. PCR reactions were performed with specific oligonucleotides. As a positive control, additional reactions were performed by using genomic DNA as template. PCR amplification with non-reverse-transcriptase-treated RNA made it possible to verify the absence of DNA contamination.
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RESULTS |
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DISCUSSION |
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Previous studies have shown that -glucuronidase activity is found in some inhabitants of the gastrointestinal tract, such as Eubacterium sp, and Bacteroides (Hawkesworth et al., 1971
; McBain & Macfarlane, 1998
; Akao, 1999
, 2000
), but up to now, no
-glucuronidase gene has been described in them. Despite the physiological importance of
-glucuronidase to human health, only the genetic elements encoding the
-glucuronidase enzymes of E. coli and L. gasseri, two subdominant intestinal bacteria, have been identified and studied (Jefferson et al., 1986
; Wilson et al., 1992
; Russel & Klaenhammer, 2001
). The discovery of this new bacterial gus gene in a predominant bacterium of the large bowel will provide a new context in which to study the effects of bacterial
-glucuronidase on gastrointestinal health and disease.
The genetic organization of the R. gnavus gus gene was surprising, as the gus gene is inserted inside genes involved in PTSs. This organization has nothing in common with any other Gram-positive or Gram-negative PTS that has been described, nor with other -glucuronidases previously studied, such as the E. coli uidA gene (Wilson et al., 1992
) or the L. gasseri gusA gene (Russel & Klaenhammer, 2001
). In E. coli, the uidA gene belongs to the uidRABC operon, in which uidR encodes the transcriptional repressor while uidB encodes the transporter, and a membrane-associated protein (uidC) is involved in the glucuronide transport system (Wilson et al., 1992
; Liang et al., 2005
). In L. gasseri, unlike in E. coli, the gusA gene is transcribed as a monocistronic unit (Russel & Klaenhammer, 2001
). The DNA surrounding the L. gasseri gusA gene does not reveal any gene encoding an obvious transport protein, indicating that L. gasseri uses either a specific transporter located elsewhere on the chromosome or an alternative transporter for glucuronide uptake (Russel & Klaenhammer, 2001
). However, in R. gnavus strain E1, the gus gene is transcribed as part of an operon of at least four ORFs. Due to this genetic organization, and taking into account the results of complementation studies in E. coli, the gus-flanking ORF2 and ORF3 are good candidates for transport proteins. ORF2 shows homology with the membrane-spanning domain IIC of the enzyme EII of the PTSs of E. coli and B. subtilis. ORF3 has two potential start codons preceded by a potential RBS. The longest product of ORF3 is a 195 amino acid protein and the shortest is a 167 amino acid protein. Both products show homology with the B. subtilis bglP product. This unusual organization of the
-glucuronidase locus suggests that this enzyme could be active on phospho-
-glucuronide. One can suppose that the gene encoding this activity has been inserted inside the PTS operon by horizontal gene transfer. However, the GC content of the gus gene, 39·9 %, does not show any divergence from the PTS genes in which it is included (GC content 37·741·8 %). The GC composition of the gus locus is not significantly lower than that of R. gnavus E1 chromosomal DNA (43 %). This trait suggests that this insertion is not a recent event or that this fragment comes from another organism having a similar GC content.
The regulation of uidA expression has been well studied in E. coli. This expression is controlled at the transcriptional level by the specific repressor UidR (Novel & Novel, 1976) and by catabolic-responsive elements (Jefferson et al., 1986
). The repressor blocks the transcription of uidABC in the absence of methylglucuronide. Expression is induced by a variety of glucuronides and is subject to catabolic repression via cAMP. In L. gasseri, the regulation of
-glucuronidase expression does not involve catabolic repression or glucuronide induction, even in the presence of a potential repressor upstream from the gene encoding the glucuronidase. In R. gnavus, the structural organization of the gus gene embedded in the IIB, IIC and IIA domains of the well-described PTS EII would suggest a co-regulation of
-glucuronidase activity by catabolic repression. As the activity was higher during the stationary phase, it is possible that activation of this gene product depends on sugar availability. As no chemically defined media were available for R. gnavus growth, it was not possible to study what kind of sugar will not induce catabolic repression in this species. In low-GC Gram-positive bacteria where catabolic repression has been described, the genes are regulated by the catabolic control protein CcpA. CcpA is a global regulator that binds to catabolic-responsive elements (CREs) in promoter regions to control carbon catabolic repression (Stülke & Hillen, 1999
). The genes regulated by CcpA share a common box upstream from the start codon called the CRE box (TGNNANCGNTNNCA). A potential box (TGAGAAGGGTAACA) was found 170 bp upstream from the ORF2 start codon, suggesting that
-glucuronidase expression is submitted to catabolic repression, as in E. coli. Because of the limited extent of the R. gnavus DNA sequence, this CRE box has not yet been described, and as neither genetic tools nor chemically defined media are available, further experiments are needed to check our hypothesis on the regulation of
-glucuronidase expression in this species. In order to obtain a better insight into the regulation of R. gnavus
-glucuronidase expression, studies are currently being carried out using the Gram-positive bacterium Lactococcus lactis, for which genetic tools and chemically defined media are available. Additionally, the gus gene can be used to construct a reporter gene vector specific to Gram-positive bacteria.
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ACKNOWLEDGEMENTS |
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Received 18 October 2004;
revised 31 March 2005;
accepted 27 April 2005.
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