Transcriptional and functional characterization of genetic elements involved in galacto-oligosaccharide utilization by Bifidobacterium breve UCC2003

Abstract

Several prebiotics, such as inulin, fructo-oligosaccharides and galacto-oligosaccharides, are widely used commercially in foods and there is convincing evidence, in particular for galacto-oligosaccharides, that prebiotics can modulate the microbiota and promote bifidobacterial growth in the intestinal tract of infants and adults. In this study we describe the identification and functional characterization of the genetic loci responsible for the transport and metabolism of purified galacto-oligosaccharides (PGOS) by Bifidobacterium breve UCC2003. We further demonstrate that an extracellular endogalactanase specified by several B. breve strains, including B. breve UCC2003, is essential for partial degradation of PGOS components with a high degree of polymerization. These partially hydrolysed PGOS components are presumed to be transported into the bifidobacterial cell via various ABC transport systems and sugar permeases where they are further degraded to galactose and glucose monomers that feed into the bifid shunt. This work significantly advances our molecular understanding of bifidobacterial PGOS metabolism and its associated genetic machinery to utilize this prebiotic.

Fig. 1

Growth profiles of B. breve strains on lactose (A) and PGOS (B). Data presented are averages of duplicate growth experiments.

Fig. 2

HPAEC-PAD analysis of post-fermentation cell-free supernatants of B. breve strains. Modified Rogosa supplemented with 0.5% GOS (AI and BI); post-fermentation supernatants of B. breve UCC2003 (AII and BII), endogalactanase positive strains: B. breve UCC2005 (AIII); NCFB2258 (AIV); NCIMB8815 (AV); NIZO658 (AVI) and UCC2008 (AVII) and endogalactanase negative strains: B. breve NCFB2257 (BIII); NCIMB11815 (BIV) and LMG13208 (BV).

Fig. 3

Schematic representation of B. breve UCC2003 gene clusters upregulated during growth of PGOS as sole carbohydrate source. The lengths of the arrows are proportional to the length of the predicted ORF and the gene locus name, which is indicative of its putative function, is indicated within the arrow. The bent arrows indicate the galC and galA promoters; the lollipop sign designates putative rho-independent terminator region. β-Galactosidase-encoding genes are indicated by blue shading, while genes encoding proteins with transport functions are shaded in yellow. Putative or proven genes encoding LacI-type transcriptional regulators are indicated by red shading.

Fig. 4

Growth profiles of B. breve UCC2003 and insertion mutant strains on ribose (AI), lactose (AII), and PGOS (AIII). Data presented are averages of duplicate growth experiments. B. β-Galactosidase activity assays of uninduced and nisin induced L. lactis cultures, NZ9000-pNZ8150, NZ9000-pNZ-lacZ2, NZ9000-pNZ-gosG, NZ9000-pNZ-galG and NZ9000-pNZ-lacZ.

Fig. 5

HPAEC-PAD analysis of post-fermentation cell-free supernatants of B. breve UCC2003 and insertion mutant strains. Modified Rogosa supplemented with 0.5% GOS (A); Post-fermentation supernatants of B. breve UCC2003 (B), UCC2003-galA (C), UCC2003-galC (D), UCC2003-galG (E), UCC2003-lacS (F), UCC2003-lacZ (G), UCC200-gosD (H), UCC2003-gosG (I). The position of lactose and galactotriose are indicated by asterisk (*) and double-asterisk (**) respectively.

Fig. 6

Mass Spectroscopy analysis of cell-free supernatants of B. breve UCC2003 (A) and B. breve UCC2003-galA (B). Samples were retained for analysis at 0, 12 and 24 h. Samples from duplicate experiments were run in triplicate and the data presented are averages of these six data sets with standard deviations.

Fig. 7

Growth profiles of (A) B. breve UCC2003 pBC1.2, UCC2003-galA-pBC1.2 and UCC2003-galA-pBC1.2-galA on lactose (Ai) and PGOS (Aii) and (B) B. breve NCFB2257-pBC1.2 and NCFB2257-pBC1.2-galA on lactose and PGOS. Data presented are averages of duplicate experiments.