To extend our understanding of bifidobacterial mutualism and carbohydrate syntrophy in the gut we adopted advanced functional genomics to create single- and double-deletion isogenic strains of the NagA encoding genes of B. breve UCC2003. The resulting strains were examined, as compared to the parent strain, for their ability to metabolise particular host derived carbohydrates. In addition, the B. breve strains were examined for their crossfeeding capability and ability to establish, in the presence of B. bifidum ATCC29521, in the gut of dam fed neonatal mice. Overall design: 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.