Combined transcriptome and proteome analysis of Bifidobacterium animalis subsp. lactis BB-12 grown on xylo-oligosaccharides and a model of their utilization

Abstract

Recent studies have demonstrated that xylo-oligosaccharides (XOS), which are classified as emerging prebiotics, selectively enhance the growth of bifidobacteria in general and of Bifidobacterium animalis subsp. lactis strains in particular. To elucidate the metabolism of XOS in the well-documented and widely used probiotic strain B. animalis subsp. lactis BB-12, a combined proteomic and transcriptomic approach was applied, involving DNA microarrays, real-time quantitative PCR (qPCR), and two-dimensional difference gel electrophoresis (2D-DIGE) analyses of samples obtained from cultures grown on either XOS or glucose. The analyses show that 9 of the 10 genes that encode proteins predicted to play a role in XOS catabolism (i.e., XOS-degrading and -metabolizing enzymes, transport proteins, and a regulatory protein) were induced by XOS at the transcriptional level, and the proteins encoded by three of these (β-d-xylosidase, sugar-binding protein, and xylose isomerase) showed higher abundance on XOS. Based on the obtained results, a model for the catabolism of XOS in BB-12 is suggested, according to which the strain utilizes an ABC (ATP-binding cassette) transport system (probably for oligosaccharides) to bind XOS on the cell surface and transport them into the cell. XOS are then degraded intracellularly through the action of xylanases and xylosidases to d-xylose, which is subsequently metabolized by the d-fructose-6-P shunt. The findings obtained in this study may have implications for the design of a synbiotic application containing BB-12 and the XOS used in the present study.

FIG. 1.

Growth of B. animalis subsp. lactis BB-12 in a rich broth containing 2% (wt/vol) glucose or XOS, respectively, as well as a control culture where no carbon source was added. Growth studies were carried out in triplicate, and a representative data set is shown. Note that without an added carbon source there is only limited growth, which is presumably due to small amounts of other carbohydrates in the complex components of the MRS. Insert: mean values of specific growth rate, maximal cell density, and pH of culture supernatants after 8 h of growth.

FIG. 2.

High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) analysis of supernatants of B. animalis subsp. lactis BB-12 cultures propagated on XOS. Concentrations of different XOS species in the supernatants of BB-12 cultures grown on XOS collected from initially grown cells and stationary cells, respectively, are designated. The results represent mean values and standard errors for results from three biological replicates.

FIG. 3.

Comparison of the protein abundance in B. animalis subsp. lactis BB-12 grown on XOS or glucose (as observed by spot patterns on a gel image). Images (from a representative 2D-DIGE gel) were obtained by differential labeling of protein samples with fluorescent dyes. Spots A (spot 18; see Fig. S2 and Table S2 in the supplemental material) and B (spots 10 to 12) correspond to sugar-binding protein (BIF_00212) and xylose isomerase (BIF_00501), respectively.

FIG. 4.

Proposed model for the catabolism of XOS in B. animalis subsp. lactis BB-12 comprising the following steps. 1, binding of XOS at the cell surface by a sugar-binding protein. 2, transport of XOS by an ABC transport system. 3, degradation of XOS to d-xylose (by a combined action of an endo-1,4-β-xylanase and a β-xylosidase). 4, conversion of d-xylose to xylulose-5-P, a key metabolite of the fructose-6-P shunt. A, endo-1,4-β-xylanase; B, β-xylosidase; C, xylose isomerase; D, xylulose kinase. The numbers indicate the respective genes in the genome of BB-12, omitting the “BIF_0.”