Human milk oligosaccharides (HMOs), which are natural bifidogenic prebiotics, were recently commercialized to fortify formula milk. However, HMO-assimilation phenotypes of bifidobacteria vary by species and strain, which has not been fully linked to strain genotype. We have recently shown that specialized uptake systems, particularly for the internalization of major HMOs (fucosyllactose (FL)), are associated with the formation of a bifidobacteria-rich gut microbial community. Phylogenetic analysis has revealed that FL transporters have diversified into two clades harboring four clusters within the Bifidobacterium genus, but the underpinning functional diversity associated with this divergence remains underexplored. In this study, we examined the HMO-consumption phenotypes of two bifidobacterial species, Bifidobacterium catenulatum subspecies kashiwanohense and Bifidobacterium pseudocatenulatum , which both possess FL binding proteins that belong to phylogenetic clusters with unknown specificities. Growth assays, heterologous gene expression experiments, and HMO-consumption analysis showed that the FL transporter type from B. catenulatum subspecies kashiwanohense JCM 15439 T conferred a novel HMO-uptake pattern that includes the complex fucosylated HMOs (lacto- N- fucopentaose II and lacto- N- difucohexaose I/II). Further genomic landscape analyses of FL transporter-positive bifidobacterial strains revealed that H-antigen or Lewis antigen-specific fucosidase gene(s) and FL transporter specificities were largely aligned. These results suggest that bifidobacteria have acquired FL transporters along with the corresponding gene sets necessary to utilize the imported HMOs. Our results provide insight into the species- and strain-dependent adaptation strategies of bifidobacteria to HMO-rich environments. Importance The gut of breastfed infants is generally dominated by health-promoting bifidobacteria. Human milk oligosaccharides (HMOs) from breastmilk selectively promote the growth of specific taxa such as bifidobacteria, thus forming an HMO-mediated, host-microbe symbiosis. While the co-evolution of humans and bifidobacteria has been proposed, the underpinning adaptive strategies employed by bifidobacteria require further research. Here, we analyzed the divergence of the critical fucosyllactose (FL) HMO transporter within Bifidobacterium . We have shown that the diversification of the solute-binding proteins of the FL-transporter led to uptake specificities of fucosylated sugars ranging from simple trisaccharides to complex hexasaccharides. This transporter and the congruent acquisition of the necessary intracellular enzymes allows for bifidobacteria to import different types of HMOs in a predictable and strain-dependent manner. These findings explain the adaptation and proliferation of bifidobacteria in the competitive and HMO-rich infant gut environment and enable accurate specificity annotation of transporters from metagenomic data.

Bifidobacteria are among the first colonizers of the infant gut, and human milk oligosaccharides (HMOs) in breastmilk are instrumental for the formation of a bifidobacteria-rich microbiota. However, little is known about the assembly of bifidobacterial communities. Here, by applying assembly theory to a community of four representative infant-gut associated Bifidobacterium species that employ varied strategies for HMO consumption, we show that arrival order and sugar consumption phenotypes significantly affected community formation. Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis, two avid HMO consumers, dominate through inhibitory priority effects. On the other hand, Bifidobacterium breve, a species with limited HMO-utilization ability, can benefit from facilitative priority effects and dominates by utilizing fucose, an HMO degradant not utilized by the other bifidobacterial species. Analysis of publicly available breastfed infant faecal metagenome data showed that the observed trends for B. breve were consistent with our in vitro data, suggesting that priority effects may have contributed to its dominance. Our study highlights the importance and history dependency of initial community assembly and its implications for the maturation trajectory of the infant gut microbiota.

The predominance of bifidobacteria in the gut of breastfed infants is attributed to the ability of these bacteria to metabolize human milk oligosaccharides (HMOs). Thus, individual HMOs such as lacto- N -tetraose (LNT) and lacto- N -neotetraose (LNnT) are considered promising prebiotics that would stimulate the growth of bifidobacteria and confer multiple health benefits to preterm and malnourished children suffering from impaired (stunted) gut microbiota development.

Bifidobacterium longum subsp. infantis (B. infantis) is a prevalent beneficial bacterium that colonizes the human neonatal gut and is uniquely adapted to efficiently use human milk oligosaccharides (HMOs) as a carbon and energy source. Multiple studies have focused on characterizing the elements of HMO utilization machinery in B. infantis; however, the regulatory mechanisms governing the expression of these catabolic pathways remain poorly understood. A bioinformatic regulon reconstruction approach used in this study implicated NagR, a transcription factor from the ROK family, as a negative global regulator of genomic loci encoding lacto-N-biose/galacto-N-biose (LNB/GNB), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT) utilization pathways in B. infantis. This conjecture was corroborated by transcriptome profiling upon nagR genetic inactivation and experimental assessment of binding of recombinant NagR to predicted DNA operators. The latter approach also implicated N-acetylglucosamine (GlcNAc), a universal intermediate of LNT and LNnT catabolism, and its phosphorylated derivatives as plausible NagR effectors. Reconstruction of NagR regulons in various Bifidobacterium lineages revealed multiple regulon expansion events, suggesting evolution from a local regulator of GlcNAc catabolism in ancestral bifidobacteria to a global regulator controlling foraging of mixtures of GlcNAc-containing host-derived glycans in mammalian gut-colonizing B. infantis and Bifidobacterium bifidum. Importance The predominance of bifidobacteria in the gut of breastfed infants is attributed to the ability of these bacteria to utilize human milk oligosaccharides (HMOs). Thus, individual HMOs such as lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) are considered promising prebiotics that would stimulate the growth of bifidobacteria and confer multiple health benefits to preterm and malnourished children suffering from impaired (stunted) gut microbiota development. However, the rational selection of HMO-based prebiotics is hampered by the incomplete knowledge of regulatory mechanisms governing HMO utilization in target bifidobacteria. This study describes NagR-mediated transcriptional regulation of LNT and LNnT utilization in Bifidobacterium longum subsp. infantis. The elucidated regulatory network appears optimally adapted to simultaneous utilization of multiple HMOs, providing a rationale to add HMO mixtures (rather than individual components) into infant formulas. The study also provides insights into the evolutionary trajectories of complex regulatory networks controlling carbohydrate metabolism in bifidobacteria.

Aim: 16S rRNA gene-based microbiota analyses (16S metagenomes) using next-generation sequencing (NGS) technologies are widely used to examine the microbial community composition in environmental samples. However, the sequencing capacity of NGS is sometimes insufficient to cover the whole microbial community, especially when analyzing soil and fecal microbiotas. This limitation may have hampered the detection of minority species that potentially affect microbiota formation and structure. Methods: We developed a simple method, termed 16S metagenome-DRIP (Deeper Resolution using an Inhibitory Primer), that not only enhances minority species detection but also increases the accuracy of their abundance estimation. The method relies on the inhibition of normal amplicon formation of the 16S rRNA gene of a target major (abundant) species during the first PCR step. The addition of a biotinylated primer that is complementary to the variable sequence of the V3-V4 region of the target species inhibits a normal amplification process to form an aberrant short amplicon. The fragment is then captured by streptavidin beads for removal from the reaction mixture, and the resulting mixture is utilized for the second PCR with barcode-tag primers. Thus, this method only requires two additional experimental procedures to the conventional 16S metagenome analysis. A proof-of-concept experiment was first conducted using a mock sample consisting of the genomes of 14 bacterial species. Then, the method was applied to infant fecal samples using a Bifidobacterium-specific inhibitory primer (n = 11). Results: As a result, the reads assigned to the family Bifidobacteriaceae decreased on average from 16,657 to 1718 per sample without affecting the total read counts (36,073 and 34,778 per sample for the conventional and DRIP methods, respectively). Furthermore, the minority species detection rate increased with neither affecting Bray-Curtis dissimilarity calculated by omitting the target Bifidobacterium species (median: 0.049) nor changing the relative abundances of the non-target species. While 115 amplicon sequence variants (ASVs) were unique to the conventional method, 208 ASVs were uniquely detected for the DRIP method. Moreover, the abundance estimation for minority species became more accurate, as revealed thorough comparison with the results of quantitative PCR analysis. Conclusion: The 16S metagenome-DRIP method serves as a useful technique to grasp a deeper and more accurate microbiota composition when combined with conventional 16S metagenome analysis methods.