Human Milk Oligosaccharide Utilization in Intestinal Bifidobacteria Is Governed by Global Transcriptional Regulator NagR

Bifidobacterium longum subsp. infantis is a prevalent beneficial bacterium that colonizes the human neonatal gut and is uniquely adapted to efficiently use human milk oligosaccharides (HMOs) as a carbon and energy source. Multiple studies have focused on characterizing the elements of HMO utilization machinery in B. longum subsp. infantis; however, the regulatory mechanisms governing the expression of these catabolic pathways remain poorly understood. A bioinformatic regulon reconstruction approach used in this study implicated NagR, a transcription factor from the ROK family, as a negative global regulator of gene clusters encoding lacto-N-biose/galacto-N-biose (LNB/GNB), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT) utilization pathways in B. longum subsp. infantis. This conjecture was corroborated by transcriptome profiling upon nagR genetic inactivation and experimental assessment of binding of recombinant NagR to predicted DNA operators. The latter approach also implicated N-acetylglucosamine (GlcNAc), a universal intermediate of LNT and LNnT catabolism, and its phosphorylated derivatives as plausible NagR transcriptional effectors. Reconstruction of NagR regulons in various Bifidobacterium lineages revealed multiple potential regulon expansion events, suggesting evolution from a local regulator of GlcNAc catabolism in ancestral bifidobacteria to a global regulator controlling the utilization of mixtures of GlcNAc-containing host glycans in B. longum subsp. infantis and Bifidobacterium bifidum.