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

O'Connell Motherway M, Kinsella M, Fitzgerald GF, van Sinderen D - Microb Biotechnol (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Alimentary Pharmabiotic Centre, National University of Ireland, Western Road, Cork, Ireland; Departments of Microbiology, National University of Ireland, Western Road, Cork, Ireland.

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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.
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fig06: 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.

Mentions: HPAEC-PAD analysis of the cell-free supernatants identified that the B. breve UCC2003-galA strain did not metabolize PGOS components with a predicted high degree of polymerization (retention time 30–48 min) (Fig. 5C). This finding was verified by Mass Spectroscopy analysis that demonstrated that B. breve UCC2003 consumed all detectable PGOS fractions, while B. breve UCC2003-galA could only metabolize PGOS fractions with a DP of up to and equal to three (Fig. 6). Interestingly, a GOS component present in fractions eluted from 22.5 to 25 min, of the HPAEC analysis, was shown to increase in the CFS of B. breve UCC2003-galC (Fig. 5D). This GOS component is likely β,1-4 linked galactotriose, which is the main product of the endogalactanase activity (Hinz et al., 2005; O'Connell Motherway et al., 2011b), and which is apparently not transported by this strain due to the galC mutation. When B. breve UCC2003-lacS was grown on PGOS, it was incapable of internalizing lactose, a GalA-mediated PGOS degradation product, which therefore accumulates in the growth medium, and which elutes at approximately 14 min under the conditions tested (Fig. 5F). The CFS post-fermentation profiles obtained following growth of UCC2003-galG (Fig. 5E), UCC2003-lacZ (Fig. 5G), or UCC2003-gosG (Fig. 5I) in PGOS-containing medium, closely resembled that of B. breve UCC2003, a result that was not unexpected given that GalG, LacZ and GosG are all intracellular β-galactosidases, while the extracellular endogalactanase activity and transport functions are fully functional in these mutant strains.


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

O'Connell Motherway M, Kinsella M, Fitzgerald GF, van Sinderen D - Microb Biotechnol (2012)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3815386&req=5

fig06: 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.
Mentions: HPAEC-PAD analysis of the cell-free supernatants identified that the B. breve UCC2003-galA strain did not metabolize PGOS components with a predicted high degree of polymerization (retention time 30–48 min) (Fig. 5C). This finding was verified by Mass Spectroscopy analysis that demonstrated that B. breve UCC2003 consumed all detectable PGOS fractions, while B. breve UCC2003-galA could only metabolize PGOS fractions with a DP of up to and equal to three (Fig. 6). Interestingly, a GOS component present in fractions eluted from 22.5 to 25 min, of the HPAEC analysis, was shown to increase in the CFS of B. breve UCC2003-galC (Fig. 5D). This GOS component is likely β,1-4 linked galactotriose, which is the main product of the endogalactanase activity (Hinz et al., 2005; O'Connell Motherway et al., 2011b), and which is apparently not transported by this strain due to the galC mutation. When B. breve UCC2003-lacS was grown on PGOS, it was incapable of internalizing lactose, a GalA-mediated PGOS degradation product, which therefore accumulates in the growth medium, and which elutes at approximately 14 min under the conditions tested (Fig. 5F). The CFS post-fermentation profiles obtained following growth of UCC2003-galG (Fig. 5E), UCC2003-lacZ (Fig. 5G), or UCC2003-gosG (Fig. 5I) in PGOS-containing medium, closely resembled that of B. breve UCC2003, a result that was not unexpected given that GalG, LacZ and GosG are all intracellular β-galactosidases, while the extracellular endogalactanase activity and transport functions are fully functional in these mutant strains.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Alimentary Pharmabiotic Centre, National University of Ireland, Western Road, Cork, Ireland; Departments of Microbiology, National University of Ireland, Western Road, Cork, Ireland.

Show MeSH