A snapshot into the uptake and utilization of potential oligosaccharide prebiotics by probiotic lactobacilli and bifidobacteria as accessed by transcriptomics, functional genomics, and recombinant protein characterization
Microorganisms that when administered in sufficient amounts exert a beneficial effect to the host are defined as probiotics. The positive clinical effects of probiotics, mainly belonging to the Bifidobacterium and Lactobacillus genera in treatments of irritated bowel disorders, gut infections and lifestyle diseases are currently well documented. Selective utilization, of primarily non-digestible carbohydrates, termed prebiotics, by probiotics has been identified as an attribute of probiotic action, however the molecular mechanisms of prebiotics utilization and in particular the specificities of carbohydrate transporters and glycoside hydrolases that confer this remain largely unknown, limiting a robust understanding for the basis of selective utilization of known prebiotics and the discovery and documentation of novel prebiotics. The aim of this Ph.D. thesis was to identify the genes involved with uptake and catabolism of potential prebiotics by the probiotics Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bl-04 as model organisms, using DNA whole genome microarrays and by in silico pathway re-construction to identify key genes for further functional analysis by gene deletions and recombinant protein characterization. Transcriptional analysis was used to measure the global gene expression, in both bacteria, grown on glucose and various prebiotics and potential prebiotics covering diverse types of glycoside linkages and compositions: ß-galacto-oligosaccharides, cellobiose, gentiobiose, isomaltose, panose, raffinose, stachyose and selected strain-specific potential prebiotics – L. acidophilus NCFM: barley ß-glucan hydrolysate, lactitol, isomaltulose and polydextrose, while for B. lactis Bl-04: maltotriose, melibiose, xylobiose and xylo-oligosaccharides were used. The differential transcriptional analysis of L. acidophilus NCFM revealed upregulation of genes encoding phosphoenolpyruvate-dependent sugar phosphotransferase systems mainly associated with disaccharide uptake, galactoside pentose hexuronide permease and ATP-binding cassette transporters were upregulated by dominantly oligosaccharides. Glycoside hydrolases from families 1, 2, 4, 13, 32, 36, 42, and 65 were found associated with the various transporters for carbohydrate catabolism. The differential transcriptional analysis of B. lactis Bl-04 identified carbohydrate transporters of the major facilitator superfamily and galactoside pentose hexuronide permeases for disaccharide uptake and ATP-binding cassette transporters mainly for uptake of oligosaccharides. These transporters were found in gene clusters with glycoside hydrolases from families 1, 2, 13, 36, 42, 43 and 77. Based on gene landscape analysis and the transcriptional findings, reconstruction of utilization pathways were done in silico. Hereafter the role of essential gene products in uptake of ß-galacto-oligosaccharides putatively facilitated by a galactoside pentose hexuronide permease and the involvement of an ATP-binding cassette transporter and an a-galactosidase for uptake of raffinose family oligosaccharides and catabolism, respectively, were confirmed by gene deletion mutants in L. acidophilus NCFM. The B. lactis Bl-04 homologous protein of the L. acidophilus NCFM raffinose specific solute binding protein displayed dual substrate specificity for raffinose family oligosaccharides and isomalto-oligosaccharides. The binding affinities (KD) to a set of a-1,6 glycosides representing both classes of ligands were in the µM range, notably lower than typical values for oligosaccharide binding to solute binding proteins. The binding was enthalpically dominated and the lower affinity owed to a large unfavorable binding entropy suggestive of a high plasticity of the ligand binding site needed to accommodate different ligands varying in size, and monosaccharide composition, but recognizing a core structure comprising an a-D-(1,6)-linked galactose or its glucose C4 epimer. Biochemical characterization of the recombinant protein validated the broad substrate specificity, however the binding affinity was 100–1000 fold lower for the preferred substrates panose and raffinose, than seen for mono-specific carbohydrate transporters previously described although any biological implication of the weaken affinities is yet to be investigated. In conclusion, differential transcriptomics revealed the global regulated gene response of L. acidophilus NCFM and B. lactis Bl-04 to potential prebiotic carbohydrates from which novel specificities for carbohydrate transporters and glycoside hydrolases were identified and validated through functional characterization. The work adds to the understanding of how probiotic bacteria can selective utilize prebiotics and how novel prebiotics can be discovered.