The porphyran degradation system is complete, phylogenetically and geographically diverse across the gut microbiota of East Asian populations

Abstract

The human gut microbiota can acquire new catabolic functions by integrating genetic material coming from the environment, for example from food-associated bacteria. An illustrative example of that is the acquisition by the human gut microbiota of Asian populations of genes coming from marine bacteria living on the surface of red algae that are incorporated into their diet when eating maki-sushi. To better understand the function and evolution of this set of algal genes corresponding to a polysaccharide utilization locus (PUL) dedicated to the degradation of porphyran, the main polysaccharide of the red algae Porphyra sp., we characterized it biochemically, assessed its genetic diversity and investigated its geographical distribution in large public worldwide datasets. We first demonstrated that both methylated and unmethylated fractions are catabolized without the help of external enzymes. By scanning the genomic data of more than 10,000 cultivated isolates as well as metagenomic data from more than 14,000 worldwide individuals, we found that the porphyran PUL is present in 17 different Phocaeicola/Bacteroides species (including 12 species that were not known to carry it), as well as in two Parabacteroides species and two genera from the Bacillota phylum, highlighting multiple lateral transfers within the gut microbiota. We then analyzed the prevalence of this porphyran PUL across 32 countries and showed that it exists in appreciable frequencies (>1%) only in East Asia (Japan, China, Korea). Finally, we identified three major PUL haplotypes which frequencies significantly differ between these East Asian countries. This geographic structure likely reflects the rate of bacterial horizontal transmission between individuals.

Fig 1. Degradation of porphyran by BpGH16C (Bacple_01703).

A) Size exclusion chromatography of the degradation products of porphyran incubated with the methyl-6O-β-porphyranase BpGH16C. The degradation profile was compared with the two P. atlantica T6c porphyranases grouped in the GH16_12 (Patl_0824) and GH16_14 (Patl_0805) sub-families. B) ¹H NMR of the disaccharides end-products of BpGH16C.

Fig 2. Crystal structure of BpGH16C (Bacple_01703).

A) Chain A of asymmetric unit is shown in cartoon representation and colored in spectrum from N- to C-terminals. The HEPES molecule in the active site is shown as stick representation. The N- and C-terminals are marked, and the strands are labeled in their order in the sequence. B) The substrate binding site in the mutant BpGH16C-E145L complexed with the D-Gal-β4-6-sulfate-L-Gal disaccharide end-product. The semitransparent surface of the protein is colored by the electrostatic potential. The residues forming the site are shown in stick representation. The sulfate group from L-galactose 6 sulfate is docked into a positively charged pocket. C) The hydrogen bonds between the disaccharide in substrate binding site residues. Several H-bonds are bridged by water molecules (W). Glu145 (in blue and thicker bonds) from the native structure is superimposed on the Leu145 in the mutants. D) Structure comparison of BpGH16C (green), PorA (PDB id-3ILF) (magenta) and PorB (PDB id- 3JUU) (yellow) showing the active site region. The single mutation of Ser129/Thr137 (PorA/PorB) (sidechains shown as spheres) to Gly132 in BpGH16C provides space for accommodating a methyl group at C6 on L-galactose residue. E) A cross-section of the surface representation of the Gal sugar and the cavity in the binding site near Gly132. There is free space within the cavity sufficient to accommodate the methyl group of a methylated porphyran.

Fig 3. Catabolism of oligo-porphyran.

¹H NMR recorded after sequential incubation of the methylated tetrasaccharide end-products of the 6-O-methyl-β-porphyranase BpGH16C with the sulfatase BpS1_11 followed by the β-L-galactosidase BpGH29 and the β-D-galactosidase BpGH2C.

Fig 4. Diversity of the porphyran PUL organization.

Gene organization of the porphyran degradation system of B. plebieus compared with other identified human gut bacteria. Phylogenetic tree was calculated using concaneted gene sequences of PUL-PorB. PUL organizations of each strain was indicated based on the available sequencing data.

Fig 5. Distribution of the PUL PorB groups in assembled metagenome.

A) Phylogenetic tree calculated with the concatenated protein sequences of PUL-PorB recorded in non-redundant human gut bacteria including isolated strains (●). The observed clades were used to create groups of homologous porphyran PUL PUL-PorB (GI, GII, GIII and GIIIrec) and divergent PUL-PorB (Outgroup). B) Distribution of the different group of the PUL-PorB in human gut metagenome assembly of South east and North East Chinese, Korea and Japanese populations.

Fig 6. Proportion of individuals with short reads mapping to the 54-nucleotides probes specific to the PUL-PorB in Asia (PUL-positive individuals), grouped by city.

Some geographic coordinates have been adjusted so that each point is visible.

Fig 7. Distribution of the various porphyran PUL among Chinese, Japanese and Korean populations.

The pie charts derive from the histogram presented in S9 Fig obtained from the analyses of short read sequencing of Chinese, Japanese and Korean metagenomics data.