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. 2013 Oct;79(20):6280-92.
doi: 10.1128/AEM.01775-13. Epub 2013 Aug 2.

Metabolism of Four α-Glycosidic Linkage-Containing Oligosaccharides by Bifidobacterium Breve UCC2003

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Metabolism of Four α-Glycosidic Linkage-Containing Oligosaccharides by Bifidobacterium Breve UCC2003

Kerry Joan O'Connell et al. Appl Environ Microbiol. .
Free PMC article

Abstract

Members of the genus Bifidobacterium are common inhabitants of the gastrointestinal tracts of humans and other mammals, where they ferment many diet-derived carbohydrates that cannot be digested by their hosts. To extend our understanding of bifidobacterial carbohydrate utilization, we investigated the molecular mechanisms by which 11 strains of Bifidobacterium breve metabolize four distinct α-glucose- and/or α-galactose-containing oligosaccharides, namely, raffinose, stachyose, melibiose, and melezitose. Here we demonstrate that all B. breve strains examined possess the ability to utilize raffinose, stachyose, and melibiose. However, the ability to metabolize melezitose was not common to all B. breve strains tested. Transcriptomic and functional genomic approaches identified a gene cluster dedicated to the metabolism of α-galactose-containing carbohydrates, while an adjacent gene cluster, dedicated to the metabolism of α-glucose-containing melezitose, was identified in strains that are able to use this carbohydrate.

Figures

Fig 1
Fig 1
Final OD600 values following 16 h of growth of various wild-type B. breve strains on 1% raffinose, 1% stachyose, 1% melibiose, or 1% melezitose. The results are mean values obtained from three separate experiments.
Fig 2
Fig 2
Comparison of the melezitose, raffinose, and stachyose gene clusters of B. breve UCC2003 with corresponding putative melezitose, raffinose, and stachyose utilization loci of other bifidobacteria. Each solid arrow represents an ORF. The length of each arrow is proportional to the length of the predicted ORF, and the gene locus name, which is indicative of its putative function, is given at the top. Orthologs are shown in the same color. The amino acid identity of each predicted protein to its equivalent protein encoded by B. breve UCC2003, expressed as a percentage, is given above each arrow.
Fig 3
Fig 3
(A) Final OD600 after 16 h of growth of UCC2003 and insertion mutants UCC2003-RafB (raffinose binding protein) and UCC2003-RafA (α-galactosidase) on 1% stachyose, raffinose, melibiose, or glucose. (B) Final OD600 following 16 h of growth of B. breve UCC2003, B. breve UCC2003-MelD [α-(1→3)-glucosidase], and B. breve UCC2003-MelA (solute binding protein) on 1% melezitose or glucose. In both panels, the results are mean values obtained from three separate experiments.
Fig 4
Fig 4
(A) HPAEC-PAD analysis indicating the breakdown of melezitose and turanose (initial concentration, 0.1 mg ml−1) by the purified recombinant protein MelD in 20 mM MOPS buffer (pH 7.0) over 24 h. The chromatogram shows results for melezitose (graph I), sucrose (graph II), and turanose (graph III) incubated with MelD. The liberation of glucose and fructose is visible as chromatographic peaks eluted at 6.25 and 5.5 min, respectively. Breakdown products are indicated by solid arrows. Chromatographic positions of carbohydrate standards are indicated by dashed arrows above the chromatogram. (B) HPAEC-PAD analysis indicating the breakdown of stachyose, raffinose, and melibiose by the purified recombinant protein RafA in 20 mM MOPS buffer (pH 7.0) over 24 h. (Graphs I and II) Stachyose (graph I) and raffinose (graph II) incubated with RafA. The liberation of galactose and sucrose is visible as chromatographic peaks eluted at 5.75 and 10.5 min, respectively. (Graph III) Melibiose incubated with RafA. The hydrolysis of this substrate to glucose and galactose is visible as a single chromatographic peak eluted at 5.75 min. Arrows are as explained for panel A.

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