Bifidobacterium longum subsp. infantis uses two different β-galactosidases for selectively degrading type-1 and type-2 human milk oligosaccharides

Abstract

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.

Bifidobacterium longum subsp. longum Exo-β-1,3-Galactanase, an Enzyme for the Degradation of Type II Arabinogalactan

“…infantis, which has 96% identity with BLLJ_0443, showed a higher specificity for ␤-1,3-Gal 2 and ␤-1,6-Gal 2 than for ␤-1,4-Gal 2 and lactose (34). Conversely, Blon_2334 (59% identity with BLLJ_1486) had high specificities for lactose and N-acetyllactosamine (35), and Blon_2123 (71% identity with BLLJ_0352) had specificity for ␤-1,4-Gal 2 (34). In addition, B. longum subsp.…”

Genomics of the Genus Bifidobacterium Reveals Species-Specific Adaptation to the Glycan-Rich Gut Environment

“…infantis, a typical fecal isolate from (breast-fed) infants, encompasses a gene cluster predicted to encode GHs and carbohydrate transporters necessary for the import and metabolism of HMOs (5). This 43-kb gene cluster encodes a variety of predicted or proven catabolic enzymes, such as fucosidases, sialidases, a ␤-hexosaminidase, and ␤-galactosidases, as well as extracellular solute binding proteins and permeases that are devoted to HMO metabolism (5,(54)(55)(56). Moreover, the genome of this microorganism contains a complete urease operon predicted to be involved in the utilization of urea, representing an important nitrogen source in milk (5).…”

Differences in the Substrate Specificities and Active-Site Structures of Two α-L-Fucosidases (Glycoside Hydrolase Family 29) fromBacteroides thetaiotaomicron

“…14) We expressed these proteins with C-terminal histidine-tags in Escherichia coli BL21 ÁlacZ (DE3). 7,15) The signal sequences were removed in both cases. The sequences of the primers used were 5 0 -ccatatgcaagaagcaaagaaggaaatt-3 0 and 5 0 -ccctcgagtgttaaagcatctcgataaat-3 0 (for BT 2970), and 5 0 -ccatatgcaacaagcaccagctccttgc-3 0 and 5 0 -ccctcgagtaatttgaaaaccggtgactg-3 0 (for BT 2192).…”

“…In this context, it is interesting to note that most -galactosidases do not liberate Gal from Lewis antigens without prior digestion by GH29-B enzymes. 7,15,16) …”