Genetic diversity of β-glucuronidase activity among 14 strains of the dominant human gut anaerobe Ruminococcus gnavus

Bacterial β-glucuronidase activity in the gut increases the enterohepatic circulation of toxic compounds and plays a major role in the etiology of colon cancer. Previously, we had found that the gus gene, which codes for β-glucuronidase in a dominant anaerobic species of the gut microbiota, Ruminococcus gnavus strain E1, is transcribed as part of an operon that includes three ORFs that code for β-glucoside permeases of the phosphotransferase systems. This genetic organization had never been described. We have now compared β-glucuronidase activity and the genetic environment of the gus gene in 14 strains of Ruminococcus gnavus.We found that five out of the seven glucuronidase-positive R. gnavus strains possessed another glucuronidase gene different from the gusA operon of R. gnavus E1. This dominant commensal intestinal species appears to have a high degree of genetic diversity in the genes that control β-glucuronidase activity.


Introduction
Glucuronidation is a major detoxification process that converts a large number of xenobiotic and endogenous substances into more hydrophilic metabolites (Tephly and Burchell,1990;Gueraud and Paris,1998).Parts of the glucuronides are secreted through the biliary route into the intestine.They are poorly reabsorbed into the bloodstream and are efficiently eliminated from the body, unless they are hydrolyzed by intestinal β-glucuronidase enzymes.However, most of the β-glucuronidase activity in the caecum and in the large intestine of rats has been attributed to bacterial enzymes (Rod and Midtvedt, 1977).β-glucuronidase activity increases the enterohepatic circulation of toxic compounds.Consequently, it plays a major role in the generation and maintenance of toxic and carcinogenic metabolites in the body, which can promote tumor formation at various sites, including the large bowel (McBain and MacFarlane,1998;Arimochi et al.,1999).We previously identified and cloned a new β-glucuronidase gene, gus, in an anaerobic strain of the dominant gut microorganism, Ruminococcus gnavus strain E1 (Beaud et al.,2005).This gus gene was transcribed as part of an operon, including three ORFs, ORF2, ORF3 and ORF5.ORF2 and ORF3 products presented strong similarities with many β-glucoside permeases of the phosphoenolpyruvate β-glucoside phosphotransferase systems (PTS), such as BglC in Escherichia coli, BglP in Bacillus subtilis and PTS enzyme II in Bacillus halodurans.ORF5 product presents strong similarities with the amino-terminal domain of Clostridium acetobutylicum β-glucosidase (bglA).Because of the significance of β-glucuronidase in numerous physiological and pathological processes, including colon cancer, we compared β-glucuronidase activity and the genetic environment of the corresponding gene in 13 R. gnavus strains and the original strain E1, isolated from human gut.

Ruminococcus gnavus strains and culture conditions
The 14 R. gnavus strains were isolated from 14 donors -(Table 1).These strains were identified at the species level on the basis of the nucleotide sequence of the DNA encoding the 16S RNA (Marcille et al., 2002).The strains were grown in an anaerobic chamber at 37 °C in reduced BHI broth (Difco) supplemented with 5g/L of yeast extract (Difco) and 5 mg/L of haemin (Sigma-Aldrich).

Enzyme assays
β-glucuronidase activity was assessed spectrophotometrically in cell extracts prepared by centrifugation of the cell pellet and disrupting it with 106 µm glass beads (Sigma-Aldrich).The enzyme activity was measured as the rate of release of para-nitro-phenol (at λ = 400nm) from para-nitro-phenyl-β-D-glucuronide (PNPG) (Sigma-Aldrich).One unit of activity is defined as 1 nmol of paranitro-phenol formed per min per milligram of total protein.Each value was calculated as the average of at least three independent determinations (Beaud et al., 2005).

Discussion
Out of the seven glucuronidase-positive R. gnavus strains, five (V42, V50, V51, V58 and V99) were found to possess a glucuronidase gene quite different from the gusA operon of R. gnavus E1.In order to be sure that these five β-glucuronidase strains did not possess a gusA homolog, lower stringency hybridizations were performed under the same conditions.The same results were obtained, so we 364 Beaud et al.  concluded that these strains have no gus gene homolog.The unexpected observation that a number of β-glucuronidasenegative R. gnavus strains possess gusA and ORF2 homologs raises further questions.Isolates of E. coli O157:H7 with nucleotide sequences for the uidA gene had no β-glucuronidase activity (Feng and Lampel,1994).This gene has a small number of mutations that result in the production of an inactive β-glucuronidase enzyme, which was still recognized by the anti-β-glucuronidase antibody.Further experiments, such as DNA sequencing and analysis, would be necessary to elucidate the true distribution of gusA and ORFs homologs in these R. gnavus strains.Her possibility could explain the preshence of gusA homologs without β-glucuronidase activity in these two strains could be the techniques used to measure the activity.We determined the in vitro β-glucuronidase activity of individual strains by disrupting a pellet of centrifuged bacterial cultures in media containing sugar but no inducer.The gus gene in R. gnavus E1 was transcribed as part of an operon that included three ORFs, ORF2, ORF3 and ORF5.The ORF2 and ORF3 products presented strong similarities with many β-glucoside permeases of the phosphoenolpyruvate β-glucoside phosphotransferase systems (PTS).
The structural organization of the gus gene suggests a regulation of β-glucuronidase activity by catabolite repression, dependent on sugar availability.As no chemically-defined medium was available for R. gnavus growth, it was not possible to study catabolite repression in this species.Further experiments with media containing inducers, such as methyl-glucuronide or bile extract, should be made with the β-glucuronidase-negative R. gnavus strains.
The true distribution of the functional gusA operon in human gut R. gnavus strains and what kind of advantage GusA expression provides to R. gnavus have yet to be determined.A number of important natural compounds, including bilirubin and endogenous hormones, are routinely excreted as glucuronide conjugates via bile into the gastrointestinal tract; consequently, R. gnavus could use glucuronides for energy production.But the natural substrates of R. gnavus are yet unknown.This information may provide clues for dietary control of β-glucuronidase activity of the intestinal microbiota.

Figure 1
Figure1β-glucuronidase activity of Ruminococcus gnavus strains, after 24 h of growth, based on the ability of cell extracts to hydrolyse PNPG.β-glucuronidase activity was expressed in nmol of degraded substrate/ min/mg of total proteins.Each value presented is the average of results from at least three independent experiments.
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Table 2 -
Summary of β-glucuronidase activity, PCR amplifications and hybridizations for each strain.All strains are Ruminococcus gnavus strains, listed in Table1.