Bifidobacterium breve UCC2003 metabolizes lacto-N-tetraose and lacto-N-neo-tetraose through overlapping, yet distinct pathways

Recent studies have begun to elucidate the mechanisms of utilisation of some human milk oligosaccharides (HMO) components by Bifidobacterium breve. However, this phenomenon is still relatively poorly understood, with little to no work to date in understanding a number of specific structures common to HMO.   In this study, we demonstrate that the prototype B. breve strain UCC2003 possesses specific metabolic pathways for the utilisation of Lacto-N-Tetraose and Lacto-N-neoTetraose, which represent the central moieties of Type I and Type II HMOs, respectively. Using a combination of experimental approaches, the enzymatic machinery involved in the metabolism of these two HMO structures was identified and characterised. Homologs of tWe also identified the key genetic loci involved in the utilisation of these HMO substrates in B. breve, B. bifidum and B. longum subsp. infantis using bioinformatic analyses were shown to be, and noted the relatively variably present among other members ofscant distribution of their homologs across the Bifidobacterium genus as a whole, withwhile noting a distinct pattern of conservation of LNB utilisation genes inamong human-associated bifidobacterial species. 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.