Bifidobacterium breve UCC2003 employs multiple transcriptional regulators in a series of glycan-induced positive feedback loops to regulate HMO metabolism

Regulation of carbohydrate metabolism in Bifidobacterium breve has been studied in detail for a variety of both plant and human-derived glycans, particularly in the model strain UCC2003. We have recently comprehensively elucidated the precise metabolic pathways by which the human milk oligosaccharide (HMO) components LNT, LNnT and LNB are utilized by B. breve UCC2003. However, no work has been carried out to date in identifying and understanding the regulatory mechanisms that control transcription of the genetic loci involved in these metabolic pathways. In the current study, we identified and characterized several transcriptional regulators involved in controlling the expression of HMO loci, and the regulatory sequences of each locus required for these regulatory mechanisms. A series of positive feedback loops inducing the expression of each locus, with the function of each regulator itself dependent on the presence of the specific monosaccharide metabolite released by the breakdown of the glycan targeted by the individual locus controlled by each respective regulator. Furthermore, we dissect the regulatory regions of each affected gene, and identify the key features within them. 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 (van Hijum et al., 2005). 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.