The bifidogenic effect of human milk oligosaccharides (HMOs) has long been known, yet the precise mechanism underlying it remains unresolved. Recent studies show that some species/subspecies of Bifidobacterium are equipped with genetic and enzymatic sets dedicated to the utilization of HMOs, and consequently they can grow on HMOs; however, the ability to metabolize HMOs has not been directly linked to the actual metabolic behavior of the bacteria. In this report, we clarify the fate of each HMO during cultivation of infant gut-associated bifidobacteria. Bifidobacterium bifidum JCM1254, Bifidobacterium longum subsp. infantis JCM1222, Bifidobacterium longum subsp. longum JCM1217, and Bifidobacterium breve JCM1192 were selected for this purpose and were grown on HMO media containing a main neutral oligosaccharide fraction. The mono-and oligosaccharides in the spent media were labeled with 2-anthranilic acid, and their concentrations were determined at various incubation times using normal phase high performance liquid chromatography. The results reflect the metabolic abilities of the respective bifidobacteria. B. bifidum used secretory glycosidases to degrade HMOs, whereas B. longum subsp. infantis assimilated all HMOs by incorporating them in their intact forms. B. longum subsp. longum and B. breve consumed lacto-N-tetraose only. Interestingly, B. bifidum left degraded HMO metabolites outside of the cell even when the cells initiate vegetative growth, which indicates that the different species/subspecies can share the produced sugars. The predominance of type 1 chains in HMOs and the preferential use of type 1 HMO by infant gut-associated bifidobacteria suggest the coevolution of the bacteria with humans.

Intracellular homeostasis for zinc is achieved through the coordinate regulation of specific transporters engaged in zinc influx, efflux, and intracellular compartmentalization. We have identified a novel mammalian zinc transporter, zinc transporter 5 (ZnT-5), by virtue of its similarity to ZRC1, a zinc transporter of Saccharomyces cerevisiae, a member of the cation diffusion facilitator family. Human ZnT-5 (hZnT-5) cDNA encodes a 765-amino acid protein with 15 predicted membranespanning domains. hZnT-5 was ubiquitously expressed in all tested human tissues and abundantly expressed in the pancreas. In the human pancreas, hZnT-5 was expressed abundantly in insulin-containing ␤ cells that contain zinc at the highest level in the body. The hZnT-5 immunoreactivity was found to be associated with secretory granules by electron microscopy. The hZnT-5-derived zinc transport activity was detected using the Golgi-enriched vesicles prepared from hZnT-5-induced HeLa/hZnT-5 cells in which exogenous hZnT-5 expression is inducible by the Tet-on gene regulation system. This activity was dependent on time, temperature, and concentration and was saturable. Moreover, zinc at a high concentration (10 mM) inhibited the growth of yeast expressing hZnT-5. These results suggest that ZnT-5 plays an important role for transporting zinc into secretory granules in pancreatic ␤ cells.

Gut microbiota of breast-fed infants are generally rich in bifidobacteria. Recent studies show that infant gut-associated bifidobacteria can assimilate human milk oligosaccharides (HMOs) specifically among the gut microbes. Nonetheless, little is known about how bifidobacterial-rich communities are shaped in the gut. Interestingly, HMOs assimilation ability is not related to the dominance of each species. Bifidobacterium longum susbp. longum and Bifidobacterium breve are commonly found as the dominant species in infant stools; however, they show limited HMOs assimilation ability in vitro. In contrast, avid in vitro HMOs consumers, Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis, are less abundant in infant stools. In this study, we observed altruistic behaviour by B. bifidum when incubated in HMOs-containing faecal cultures. Four B. bifidum strains, all of which contained complete sets of HMO-degrading genes, commonly left HMOs degradants unconsumed during in vitro growth. These strains stimulated the growth of other Bifidobacterium species when added to faecal cultures supplemented with HMOs, thereby increasing the prevalence of bifidobacteria in faecal communities. Enhanced HMOs consumption by B. bifidum-supplemented cultures was also observed. We also determined the complete genome sequences of B. bifidum strains JCM7004 and TMC3115. Our results suggest B. bifidum-mediated cross-feeding of HMOs degradants within bifidobacterial communities.

The human gut microbiota established during infancy has persistent effects on health. In vitro studies have suggested that human milk oligosaccharides (HMOs) in breast milk promote the formation of a bifidobacteria-rich microbiota in infant guts; however, the underlying molecular mechanism remains elusive. Here, we characterized two functionally distinct but overlapping fucosyllactose transporters (FL transporter-1 and -2) from Bifidobacterium longum subspecies infantis. Fecal DNA and HMO consumption analyses, combined with deposited metagenome data mining, revealed that FL transporter-2 is primarily associated with the bifidobacteria-rich microbiota formation in breast-fed infant guts. Structural analyses of the solute-binding protein (SBP) of FL transporter-2 complexed with 2′-fucosyllactose and 3-fucosyllactose, together with phylogenetic analysis of SBP homologs of both FL transporters, highlight a unique adaptation strategy of Bifidobacterium to HMOs, in which the gain-of-function mutations enable FL transporter-2 to efficiently capture major fucosylated HMOs. Our results provide a molecular insight into HMO-mediated symbiosis and coevolution between bifidobacteria and humans.

The breast-fed infant intestine is often colonized by particular bifidobacteria, and human milk oligosaccharides (HMOs) are considered to be bifidogenic. Recent studies showed that Bifidobacterium longum subsp. infantis can grow on HMOs as the sole carbon source. This ability has been ascribed to the presence of a gene cluster (HMO cluster-1) contained in its genome. However, the metabolism of HMOs by the organism remains unresolved because no enzymatic studies have been completed. In the present study, we characterized β-galactosidases of this subspecies to understand how the organism degrades type-1 (Galβ1-3GlcNAc) and type-2 (Galβ1-4GlcNAc) isomers of HMOs. The results revealed that the locus tag Blon_2016 gene, which is distantly located from the HMO cluster-1, encodes a novel β-galactosidase (Bga42A) with a significantly higher specificity for lacto-N-tetraose (LNT; Galβ1-3GlcNAcβ1-3Galβ1-4Glc) than for lacto-N-biose I (Galβ1-3GlcNAc), lactose (Lac) and type-2 HMOs. The proposed name of Bga42A is LNT β-1,3-galactosidase. The Blon_2334 gene (Bga2A) located within the HMO cluster-1 encodes a β-galactosidase specific for Lac and type-2 HMOs. Real-time quantitative reverse transcription-polymerase chain reaction analysis revealed the physiological significance of Bga42A and Bga2A in HMO metabolism. The organism therefore uses two different β-galactosidases to selectively degrade type-1 and type-2 HMOs. Despite the quite rare occurrence in nature of β-galactosidases acting on type-1 chains, the close homologs of Bga42A were present in the genomes of infant-gut associated bifidobacteria that are known to consume LNT. The predominance of type-1 chains in HMOs and the conservation of Bga42A homologs suggest the coevolution of these bifidobacteria with humans.