A metagenomic β-glucuronidase uncovers a core adaptive function of the human intestinal microbiome

Abstract

In the human gastrointestinal tract, bacterial β-D-glucuronidases (BG; E.C. 3.2.1.31) are involved both in xenobiotic metabolism and in some of the beneficial effects of dietary compounds. Despite their biological significance, investigations are hampered by the fact that only a few BGs have so far been studied. A functional metagenomic approach was therefore performed on intestinal metagenomic libraries using chromogenic glucuronides as probes. Using this strategy, 19 positive metagenomic clones were identified but only one exhibited strong β-D-glucuronidase activity when subcloned into an expression vector. The cloned gene encoded a β-D-glucuronidase (called H11G11-BG) that had distant amino acid sequence homologies and an additional C terminus domain compared with known β-D-glucuronidases. Fifteen homologs were identified in public bacterial genome databases (38-57% identity with H11G11-BG) in the Firmicutes phylum. The genomes identified derived from strains from Ruminococcaceae, Lachnospiraceae, and Clostridiaceae. The genetic context diversity, with closely related symporters and gene duplication, argued for functional diversity and contribution to adaptive mechanisms. In contrast to the previously known β-D-glucuronidases, this previously undescribed type was present in the published microbiome of each healthy adult/child investigated (n = 11) and was specific to the human gut ecosystem. In conclusion, our functional metagenomic approach revealed a class of BGs that may be part of a functional core specifically evolved to adapt to the human gut environment with major health implications. We propose consensus motifs for this unique Firmicutes β-D-glucuronidase subfamily and for the glycosyl hydrolase family 2.

Fig. 1.

Screening of BG activity from metagenomic libraries. The metagenomic clones originated from different libraries derived from healthy and Crohn's disease subjects’ feces and from a distal ileum biopsy. Metagenomic libraries were screened for over-expressing clones (compared with the uidA⁺ E. coli receiving strain) using PNP-G as substrate with a two-step method: a visual detection and a quantitative validation. Fosmid activity was further assessed by transfer into a BG-negative strain (E. coli L90 ΔuidA). Subclones (3-kbp fragments inserted into pcDNA 2.1 plasmids) were further screened for PNP-G deglucuronidation, and candidate genes were cloned into an expression vector (pRSFDuet-1) to definitively confirm BG activity. Random screening of 4,608 metagenomic clones from fecal and ileal libraries (E. coli DH10B as receiving strain) involved the following: (A) a qualitative screen with precocity of β-glucuronidase activity detection that yielded 1.79% of clones over-expressing the activity; (B) a quantitative validation of clones over-expressing the activity (E. coli DH10B deprived of insert as control) that yielded 0.73% of positive clones; (C) a selection of fosmids generating β-glucuronidase activity (following transformation of a β-glucuronidase-negative strain) yielding 0.41% of positive clones among all clones tested; and (D) the identification of the genes involved (subcloning, sequencing, and bioinformatic analysis).

Fig. 2.

BG activity of positive metagenomic fosmids in E. coli DH10B and E. coli L90 ΔuidA. Activity was assayed using PNP-G as substrate and expressed in units (U) of BG activity (ΔOD 405 nm·min⁻¹ · mg⁻¹). Means and SD values are determined from experiments performed in quadruplicates for each clone.

Fig. 3.

Amino acid sequence alignment of the unique BG (H11G11-BG) and known BGs from the gut microbiota. Alignment was performed using the ClustalW multiple sequence alignment program. Framed amino acids were conserved in at least three sequences.

Fig. 4.

Identification of a unique class of β-D-glucuronidase, phylogenetic affiliation, and association with potential symporters. Detailed tree view of the H11G11BG-like homologs identified in GenBank genomic databases (Fig. S1). The unique BGs (>50% of both coverage and similarity with H11G11-BG) have been identified only in Firmicutes species, but a potential BG subgroup has also been identified in Bacteroidetes constituting an independent subclass of BGs. Strains known to generate a BG activity are F. prausnitzii M21/2, R. gnavus ATCC 29149, and B. capillosus ATCC 29799. Strains demonstrated as BG positive in this study are S. variabile DSM 15176, B. formatexigens DSM 14469, C. bartlettii DSM 16795, B. ovatus ATCC 8483, P. merdae ATCC 43184, and P. johnsonii DSM 18315. ^aClassification according to Carlier et al. (23). ^bPhylogenetic assignation of metagenomic inserts are as described in Materials and Methods (H11G11 insert: 41% Clostridium genus. C7D2 insert: 81% R. gnavus species.).

Fig. 5.

Conserved motifs within the H11G11-BG group. (A) Conserved motifs within the 17 proteins identified in this study (>50% coverage and similarity with H11G11-BG). (B) Signatures proposed for the unique Firmicutes BGs. (C) Previously undescribed glycosyl hydrolase family 2 patterns, including the 17 unique Firmicutes BGs identified in this study and the three Bacteroidetes BGs. Amino acids added compared to the actual patterns are in blue.

Fig. 6.

Frequency of the unique β-glucuronidases and known β-glucuronidases in environmental and human gut metagenomes. Homologs of unique and known BG proteins were searched in NCBI metagenomic project datasets (Materials and Methods). The hit threshold was at least 50% similarity with 50% sequence coverage. Results of frequencies were expressed as hits per base pair to correct the different sizes of the metagenomic datasets investigated.

Fig. 7.

Distribution of the unique and known BGs among adult, child, and infant gut metagenomes. Homologs of unique and known BG proteins were searched in the two projects Human Gut Metagenome (13 healthy individuals) (ID 28117) and Human Distal Gut Biome (ID16729) as described in Fig. 6. Results are presented per BG group (i.e., Firmicutes-BGs, Bacteroidetes-BGs, UidA homologs) and expressed as means of hits per base pair for each group (details per BG protein, Fig. S6).