Transcriptional and Functional Characterization of genetic elements involved in galacto-oligosaccharide utilisation by Bifidobacterium breve UCC2003

It is increasingly recognised that the gastrointestinal microbiota plays a critical role in human health and promising evidence is accumulating that with dietary strategies, of prebiotic intervention, microbiota imbalances can be corrected and host health improved. Several prebiotics are widely used commercially in foods including inulin, fructo-oligosaccharides, galacto-oligosaccharides and resistant starches and there is convincing evidence, in particular for galacto-oligosaccharides, that prebiotics can modulate the microbiota and promote the growth of bifidobacteria in the intestinal tract of infants and adults. In this study we describe the identification and functional characterisation of the genetic loci responsible for the transport and metabolism of purified galacto-oligosaccharides (PGOS) by our model bifidobacterial strain, B. breve UCC2003. We further demonstrate that the extracellular endogalactanase specified by several B. breve strains, including B. breve UCC2003, is essential for metabolism of PGOS components with a long retention time and high degree of polymerisation. These PGOS components are transported into the bifidobacterial cell via various ABC transport systems and sugar permeases where they are further metabolised to galactose and glucose monomers that feed into the bifid shunt. This research described here advances our understanding of GOS metabolism by bifidobacteria and for the future there is great potential for exploiting bifidobacterial beta-galactosidase to create targeted prebiotics that can enrich for selected Bifiobacteria sp. and other beneficial microbes among the gut microbiota. DNA-microarrays containing oligonucleotide primers representing each of the 1864 annotated genes on the genome of B. breve UCC2003 were designed by and obtained from Agilent Technologies (Palo Alto, Ca., USA). Methods for cell disruption, RNA isolation, RNA quality control, complementary DNA synthesis and labeling were performed as described previously (Pokusaeva et al., 2009). Labeled cDNA was hybridized using the Agilent Gene Expression hybridization kit (part number 5188-5242) as described in the Agilent Two-Color Microarray-Based Gene Expression Analysis v4.0 manual (G4140-90050). Following hybridization, microarrays were washed in accordance with Agilent’s standard procedures and scanned using an Agilent DNA microarray scanner (model G2565A). Generated scans were converted to data files with Agilent's Feature Extraction software (Version 9.5). DNA-microarray data were processed as previously described (Garcia De La Nava et al., 2003). Differential expression tests were performed with the Cyber-T implementation of a variant of the t-test (Long et al., 2001). A gene was considered differentially expressed when p < 0.001 and an expression ratio of >3 or <0.33 relative to the control.