Project description:Bifidobacterium longum subsp. infantis is a bacterial commensal that colonizes the breast-fed infant gut where it utilizes indigestible components delivered in human milk. Accordingly, human milk contains several non-protein nitrogenous molecules, including urea at high abundance. This project investigates the degree to which urea is utilized as a primary nitrogen source by Bifidobacterium longum subsp. infantis and incorporation of hydrolysis products into the expressed proteome.
Project description:Human milk oligosaccharides (HMOs) function as prebiotics for beneficial bacteria in the developing gut, often dominated by Bifidobacterium spp. To understand the relationship between Bifidobacterium utilizing HMOs and how the metabolites that are produced could affect the host, we analyzed the metabolism of HMO 2’-fucosyllactose (2’-FL) in Bifidobacterium longum ssp. infantis Bi-26. RNA-seq and metabolite analysis (NMR/GCMS) was performed on samples at early (A600=0.25), mid-log (0.5-0.7) and late-log phases (1.0-2.0) of growth. Transcriptomic analysis revealed many gene clusters including three novel ABC-type sugar transport clusters to be upregulated in Bi-26 involved in processing of 2’-FL along with metabolism of its monomers glucose, fucose and galactose. Metabolite data confirmed the production of formate, acetate, 1,2-propanediol, lactate and cleaving of fucose from 2’-FL. The formation of acetate, formate, and lactate showed how the cell uses metabolites during fermentation to produce higher levels of ATP (mid-log compared to other stages) or generate cofactors to balance redox. We concluded 2’-FL metabolism is a complex process involving gene clusters throughout the genome producing more metabolites compared to lactose. These results provide valuable insight on the mode-of-action of 2’-FL utilization by Bifidobacterium longum ssp. infantis Bi-26.
Project description:Human milk oligosaccharides (HMOs) function as prebiotics for beneficial bacteria in the developing gut, often dominated by Bifidobacterium spp. To understand the relationship between Bifidobacterium utilizing HMOs and how the metabolites that are produced could affect the host, we analyzed the metabolism of HMO 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3FL and difucosyllactose (DFL) in Bifidobacterium longum ssp. infantis Bi-26 and ATCC15697. RNA-seq and metabolite analysis was performed on samples at early (A600=0.25), mid-log (0.5-0.7) and late-log phases (1.0-2.0) of growth.
Project description:The purpose of this project was to determine the whole transcriptome response of Bifidobacterium longum subsp. infantis to human milk urea compared to complex nitrogen and L-cysteine.
Project description:The purpose of this project was to determine the whole transcriptome response of Bifidobacterium longum subsp. Infantis to pooled and individual human milk oligosaccharides (HMO) relative to lactose
Project description:Diet-microbe interactions play a crucial role in infant development and modulation of the early-life microbiota. The genus Bifidobacterium dominates the breast-fed infant gut, with strains of B. longum subsp. longum (B. longum) and B. longum subsp. infantis (B. infantis) particularly prevalent within the early-life microbiota. Although, transition from milk to a more diversified diet later in infancy initiates a shift to a more complex microbiome, with concurrent reductions in Bifidobacterium abundance, specific strains of B. longum may persist in individual hosts for prolonged periods of time. Here, we sought to investigate the adaptation of B. longum to the changing infant diet during the early-life developmental window. Genomic characterisation of 75 strains isolated from nine either exclusively breast- or formula-fed infants in the first 18 months of their lives revealed subspecies- and strain-specific intra-individual genomic diversity with respect to glycosyl hydrolase families and enzymes, which corresponded to different dietary stages. Complementary phenotypic growth studies indicated strain-specific differences in human milk oligosaccharide and plant carbohydrate utilisation profiles between and within individual infants, while proteomic profiling identified proteins involved in metabolism of selected carbohydrates. Our results indicate a strong link between infant diet and B. longum subspecies/strain genomic and carbohydrate utilisation diversity, which aligns with a changing nutritional environment i.e. moving from breast milk to a solid food diet. These data provide additional insights into possible mechanisms responsible for the competitive advantage of this bifidobacterial species and their long-term persistence in a single host and may contribute to rational development of new dietary therapies for this important development window.
Project description:Members of the serpin (serine protease inhibitor) superfamily have been identified in higher, multicellular eukaryotes, as well as in bacteria, although surveillance of available genome sequences indicates that bacterial serpin-encoding (ser) homologs are not widely distributed. In members of the genus Bifidobacterium this gene appears to be present in at least five, and perhaps up to nine, out of 30 species tested. Moreover, phylogenetic analysis using available bacterial and eukaryotic serpin sequences revealed that bifidobacteria specify serpins that form a separate clade. We characterized the ser210B locus of Bifidobacterium breve 210B, which consists of a number of genes, whose deduced protein products display significant similarity to proteins encoded by corresponding loci found in several other bifidobacteria. Northern hybridization, primer extension, micro array analysis, RT-PCR and Quantitative Real Time (qRT) - PCR analysis revealed that a 3.5 kb polycistronic mRNA, encompassing the ser210B operon with a single transcriptional start site, is strongly induced following treatment of B. breve 210B cultures with particular proteases. In contrast, transcription of the ser homolog of other bifidobacteria, such as Bifidobacterium longum subsp. infantis, Bifidobacterium dentium and B. longum subsp. longum, appears to be triggered by a different set of proteases Transcriptional response to protease treatments (kallikrein, papain and chymotrypsin) of Bifidobacterium breve 210B
Project description:Human milk oligosaccharides (HMOs) are highly diverse complex carbohydrates secreted in human milk. HMOs are indigestible by the infant and instead are metabolized by bifidobacteria in the infant gut microbiome to produce molecules that promote infant health and development. 2´fucosyllactose (2´FL) is an abundant HMO and is utilized by Bifidobacterium longum subsp. infantis, a predominant member of the infant gut microbiome. Currently, there is not a scientific consensus on how or if bifidobacteria metabolize the fucose portion of 2´FL or free fucose. This proteomic analysis was conducted in order to characterize the metabolic pathway by which B. infantis utilizes fucose.
Project description:Bifidobacterium longum subsp. infantis (B. 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. 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 genomic loci encoding lacto-N-biose/galacto-N-biose (LNB/GNB), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT) utilization pathways in B. 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 effectors. Reconstruction of NagR regulons in various Bifidobacterium lineages revealed multiple regulon expansion events, suggesting evolution from a local regulator of GlcNAc catabolism in ancestral bifidobacteria to a global regulator controlling foraging of mixtures of GlcNAc-containing host-derived glycans in mammalian gut-colonizing B. infantis and Bifidobacterium bifidum.
Project description:Members of the serpin (serine protease inhibitor) superfamily have been identified in higher, multicellular eukaryotes, as well as in bacteria, although surveillance of available genome sequences indicates that bacterial serpin-encoding (ser) homologs are not widely distributed. In members of the genus Bifidobacterium this gene appears to be present in at least five, and perhaps up to nine, out of 30 species tested. Moreover, phylogenetic analysis using available bacterial and eukaryotic serpin sequences revealed that bifidobacteria specify serpins that form a separate clade. We characterized the ser210B locus of Bifidobacterium breve 210B, which consists of a number of genes, whose deduced protein products display significant similarity to proteins encoded by corresponding loci found in several other bifidobacteria. Northern hybridization, primer extension, micro array analysis, RT-PCR and Quantitative Real Time (qRT) - PCR analysis revealed that a 3.5 kb polycistronic mRNA, encompassing the ser210B operon with a single transcriptional start site, is strongly induced following treatment of B. breve 210B cultures with particular proteases. In contrast, transcription of the ser homolog of other bifidobacteria, such as Bifidobacterium longum subsp. infantis, Bifidobacterium dentium and B. longum subsp. longum, appears to be triggered by a different set of proteases